Background
Type B Coxsackieviruses (CV-B) are common encountered pathogens that, although mostly limited to asymptomatic and subclinical infections, are known for their wide tropism and for their broad spectrum of associated diseases (
reviewed in [
1]). Indeed, when the infection is symptomatic, it is generally localized to the gastrointestinal tract (the primary site of replication for those enteric viruses), and more rarely to the oropharynx. When virus replication persists despite the immune response, the virus reaches the blood circulation through mesenteric lymph nodes, then several target tissues such as heart, pancreas, spleen, liver, spinal cord, etc. Indeed, CV-B have been associated to several acute (meningitis, myocarditis, pancreatitis, encephalitis) and chronic diseases (chronic myocarditis, dilated cardiomyopathy, type 1 diabetes) that are often severe, even life-threatening, particularly in newborns and young children, thus constituting a serious public health problem [
1‐
3].
The six CV-B serotypes (CV-B1 to 6) belong to the
Enterovirus B species, from the
Enterovirus genus (actually encompassing at least 271 human serotypes distributed in 7 species), of the
Picornaviridae family [
4,
5]. They are small, non-enveloped, icosahedral, positive-sense single-stranded RNA viruses.
Due to their resistance in the environment, CV-B are essentially transmitted through the fecal-oral mode, and occasionally through the respiratory route [
6]. The high frequency of CV-B infections among neonates however suggests a possible vertical transmission of those viruses, at least in some cases [
3,
7]. Several epidemiological, serological and virological arguments are in favor of this hypothesis. Indeed, increased levels of anti-CV-B antibodies have been found in pregnant women in association with an infection of the offspring [
8,
9]. The viral genome has also been detected in maternal and offspring tissues [
2,
9,
10]. Vertical transmission of CV-B may occur either in utero (antenatally) through the transplacental way [
11], or perinatally during delivery [
9].
CV-B vertical transmission has been associated to an elevated risk of abortion [
8,
10,
12‐
15] and stillbirth [
16,
17]. In the case of live birth, vertically transmitted CV-B seem largely involved in many life-threatening diseases affecting fetuses, newborns and young infants [
2,
3,
7,
18,
19]. On the basis of the presence of a viremia or the appearance of clinical symptoms, about 22% of fatal CV-B infections of the neonates, result from an intra-uterine infection [
7]. Moreover, maternal CV-B infections during pregnancy would predispose offspring to the development of autoimmune diseases such as type 1 diabetes [
20]. Infections with CV-B during pregnancy are however generally neglected compared to those by other pathogens such as rubella virus, Zika virus,
Toxoplasma, etc.….
Considering the frequency of that mode of contamination by CV-B, the width and the severity of its consequences, CV-B vertical transmission deserves further investigation, in an attempt to develop preventive and/or therapeutic strategies. In this context, our current study aims to better explore CV-B4 vertical transmission using a mouse model.
Discussion
Vertical transmission is a way of contamination by CV-B that, despite constituting a serious problem, as explained above, is not sufficiently recognized and not thoroughly investigated. Most of our current knowledge on this subject comes from clinical observations in humans. Few investigations on CV-B vertical transmission were carried on mice [
22,
28‐
33], an experimental tool however frequently used to explore various aspects of infection by those viruses, difficult to address in humans. Different viral and mice strains were used, together with different inoculation periods and routes, as well as different methods of analysis. Altogether, those studies generated numerous data, but the issue of CV-B vertical transmission is not totally elucidated and disserves further work. It is in this context that joins our current study.
As in a previous study [
26], we performed our experiments with the outbred
Swiss albino mouse strain for a better representation of the heterogeneity inside the human population. We equally used the viral strain CV-B4 E2 that revealed to target numerous tissues.
The time of inoculation during pregnancy was an important point to consider since, according to previous studies, it seems to highly influence the outcome of CV-B infection in offspring [
22,
28,
31‐
33]. Indeed, Dalldorf and Gifford [
28], who were the first to investigate CV-B vertical transmission in mice, noticed that CV-B1 pancreatic line intraperitoneally inoculated to mice of the
Albany Standard strain in the third week of gestation, resulted in an increase in the morbidity/mortality rate among offspring (from 20 to 43% in the first and second week, respectively, to 77% in the third week), and thus to an increase in the severity of the infection at that stage. Conversely, Lansdown [
31] reported that intramuscular CV-B3 inoculation of
Swiss mice during the first week of gestation (day 4G or 8G) induces more pregnancy loss than an inoculation of the same virus during the second week (day 12G). Here, fetal wastage was attributed to the nutritive deficit resulting from destruction of the maternal exocrine pancreas by the virus. By the same, in the investigation carried by Modlin and Crumpacker [
32] with outbred CD-1 mice, oral inoculation with CV-B1 in the first week of gestation (day 7G), despite causing a less severe infection in dams, induced significantly more abortions than inoculation in the third week (day 14G and 16G). In another investigation, also carried on CD-1 mice, maternal oral inoculation by CV-B4 E2 at day 4G or day 17G had little effect on pregnancy outcome, whereas infection at day 10G affected dams and/or offspring [
22]. In that same study, inoculation at day 17G predisposed to an aggravation of the consequences (severe pancreatic inflammations and hyperglycemia) of a post-natal challenge of pups by the same viral strain [
22]. That difference in susceptibility to infection along the different gestational periods was attributed to physiological changes in hormonal rates that would be associated to a decrease in immunity [
28,
32]. Variations in the level of expression of the Coxsackie/Adenovirus receptor (CAR) protein, which revealed to be an essential molecule for the embryonic development [
34,
35], can also explain that difference in susceptibility to CV-B infection during the different stages of gestation. Being inspired by the results of Bopegamage et al., [
22] working with the same viral strain as well as outbred mice, we chose to inoculate our mice at day 10G (second week) and 17G (third week of gestation).
As illustrated in the
Results section, inoculation at day 10G, but not at day 17G, was followed by a significant weight loss in pregnant dams associated to an important rate of abortion and a reduced number of offspring per litter, which is in agreement with what has been observed previously [
22,
28]. A reduction in the mean body weight of newborn pups was also noticed following virus inoculation at day 10G (data not shown) which is reminiscent of what was previously reported in fetuses sampled at the end of pregnancy following maternal inoculation at day 8G [
31,
36]. Weight loss could just reflect morbidity among infected animals or at least changes on their state of health.
Such consequences were not observed following inoculation at day 17G, maybe because the infection occurs too late during pregnancy to affect its outcome. Inoculation at that gestational stage seems however to have more effect on dams, here manifesting through premature delivery, then unusual behavior (possibly because delivery occurs during the acute phase of the infection). An increase in susceptibility of dams to CV-B infection with advancing pregnancy has already been described by other teams [
28,
32]. Cannibalism (destruction of litters by their mothers) at birth has equally been reported in one of those studies together with evident morbidity of dams for at least 1 week postpartum [
28].
No delay in the fetal growth could however been evidenced as previously reported by others [
36,
37], whatever was the inoculation period. By the same, morbidity among offspring manifested only in two cases in the current investigation (one case of paralysis, and one case of pancreatitis), which is negligible if we consider the total number of examined pups, and reminiscent of the results of Bopegamage et al., [
22] that observed normal histology and normal blood glucose levels in offspring born to CV-B4 E2-inoculated dams.
In order to document CV-B4 E2 infection, we began with a rather simplistic approach, namely the detection of anti-CV-B4 antibodies by seroneutralization. Indeed, numerous studies documented a neutralizing response in CV-B-inoculated mice, a response that, as in humans, can be considered as an indirect proof of infection [
24,
38,
39]. It has been previously reported that 90% of pregnant CD-1 outbred
Swiss mice orally inoculated with CV-B3, late in pregnancy, developed IgG antibodies to CV-B3 starting from 5 days p.i. [
33]. Those antibodies seemed to protect offspring against postnatal mortality, but not against stillbirth. To the best of our knowledge, only two studies already reported maternal transfer of anti-CV-B antibodies to offspring in mice, and the effect of such antibodies seems rather contradictory and disserves further investigation [
22,
40]. Indeed, passively transferred maternal antibodies enhanced the infection of offspring during challenge in the investigation of Bopegamage et al., [
22], whereas they protected challenged offspring from infection in the work of Larsson et al., [
40]. In our current work, anti-CV-B4 antibodies were retrieved in fetuses (day 17G) and, even after birth, roughly maintained at lower levels than in dams, which suggests a partial transplacental transfer (as strengthened by antibodies detection in the placenta) of maternal antibodies. The progressive increase in the number of seropositive pups, then their decrease after day 21, let us equally think about an additional transfer via breastfeeding, rather than a
de novo synthesis by those too young animals. Indeed, the human milk was shown to contain anti-enterovirus antibodies that can neutralize the virus in vitro [
41] and protect newborns from infection [
42]. Although not a common diagnostic strategy, the detection of anti-CV-B5 antibodies in the amniotic fluid has been reported [
43], hence the idea of including such sample in our analysis that, all the same, gave negative results. Although maternally transferred neutralizing antibodies could be responsible for the protection of our pups from morbidity and mortality, they did not prevent vertical transmission of the virus to them, as discussed below.
Considering the fact that the virus needs 1 to 2 days to reach fetuses [
29,
30,
32] and that offspring born to dams inoculated 1 day before delivery escape from infection [
28], only tissues from offspring delivered at day 19G and day 20G were considered in the analysis for virus infection following inoculation at day 17G.
Here we investigated CV-B4 vertical transmission by two complementary approaches, viral RNA detection and viral progeny titration (to evaluate virus replication). Both methods were concordant since revealing the same high proportion of infected offspring (18/18 and 11/12 following inoculation at day 10G and day 17G, respectively, details not shown in the results of progeny titration). In a previous study with CD-1 mice orally inoculated with CV-B3, virus could be recovered from fetal tissue in only a small percentage (3 to 13%) of pregnancies [
33].
CAR is highly expressed in several fetal organs, which explains the increased susceptibility of fetuses, but equally of pregnant dams, to CV-B infections [
15,
34,
35]. Several tissues can be targeted during in utero CV-B4 Infection.
Virus detection in fetuses is a direct proof of antenatal in utero virus transmission. This is supported by virus detection in key tissues in vertical transmission (uterus, amniotic sac, amniotic fluid, placenta and umbilical cord). Otherwise, perinatal transmission during delivery cannot be excluded and is often supported by the reincrease of viral titers after birth.
Infection of dams’ uteri was evidenced by viral RNA detection and virus isolation in the current study, and virus isolation in a previous one [
32]. An investigation outlining the involvement of CAR in the susceptibility to CV-B3 in ICR mice, showed that CAR is highly expressed in the epithelium and glands of the uterine endometrium [
15], thus, making the uterus a privileged target for CV-B, as observed in the investigation of Modlin & Crumpacker [
32].
Virus detection in the amniotic fluid has already been reported in humans [
2], but the current investigation gives for the first time an evidence of infection of the mouse amniotic sac and amniotic fluid.
Virus detection in the placenta and the umbilical cord, and at levels comparable to those in fetal tissues, strengthens in utero virus transmission through the transplacental way. Indeed, it was outlined that the placenta is a target for CV-B3 and CV-B4 [
29,
30,
32,
33]. The failure of detecting the virus 7 days p.i. in some samples is not so intriguing since it has already been reported that virus infects the placenta 1 to 2 days after maternal infection but persists at important levels only 3 to 4 days [
32].
CV-B4 E2 detection in offspring’s pancreas seems rather evident for that so-called diabetogenic strain that is well known for its pancreotropism [
44].
Viral RNA and progeny detection in offspring’s hearts is equally not surprising since, in addition to be a main target of CV-B4, the fetal heart highly expresses CAR. That latest plays an essential role in early cardiac development and regulates cardiac remodeling in the embryo [
35,
45]. Indeed, CAR-knock-out mice die in the 11th gestational day due to cardiac anomalies [
35,
46].
Our experimental model is the first to describe virus persistence following vertical transmission, since viral RNA could be detected until 50 and 70 days postpartum in the heart and the pancreas, respectively. In previous studies, CV-B3 [
29] and CV-B4 [
30] inoculated during the third week of pregnancy, were found in fetal tissues for a period that never exceeded 3 to 4 days. In the investigation by Bopegamage et al., [
22], who equally used RT-PCR, no trace of infection was evident 30 days postpartum and authors did not search for the virus before that time point (what let them think that even vertical transmission did not occur). Virus persistence is considered as one of the main mechanisms leading to the development of chronic diseases associated to CV-B infections.