RESEARCH ARTICLE

Outcome of challenge with coxsackievirus B4 in young mice after maternal infection with the same virus during gestation Shubhada Bopegamage1, Jana Precechtelova1, Lenka Marosova1, Darina Stipalova1, Martin Sojka1, Maria Borsanyiova1, Pavol Gomolcak2, Katarina Berakova2 & Jochem M. D. Galama3 Enterovirus Laboratory, Virology Department, Slovak Medical University, Bratislava, Slovak Republic; 2Cytopathos s. r. o., Bratislava, Slovak Republic; and 3Virology Section, Department of Medical Microbiology, Radboud University Medical Center, Nijmegen, the Netherlands

IMMUNOLOGY & MEDICAL MICROBIOLOGY

1

Correspondence: Shubhada Bopegamage, Limbova 12, Bratislava 83303, Slovak Republic. Tel.: +4 212 59370777; fax: +4 212 59370683; e-mail: [email protected] Received 1 July 2011; revised 30 September 2011; accepted 10 October 2011. Final version published online 30 November 2011. DOI: 10.1111/j.1574-695X.2011.00886.x Editor: Alfredo Garzino-Demo Keywords coxsackievirus B4; oral route of infection; mice; gestation; challenge of offspring.

Abstract Enteroviral infections go usually unnoticed, even during pregnancy, yet some case histories and mouse experiments indicate that these viruses may be transmitted vertically. More frequently, however, transmission occurs by (fecal) contamination during and shortly after birth. The aim of this study was to investigate the effect of maternal infection in mice (1) on gravidity outcome and (2) on subsequent challenge of the offspring with the same virus. CD1 outbred female mice were infected by the oral route with coxsackievirus B4 strain E2 or mock-infected at days 4, 10, or 17 of gestation. Weight and signs of sickness were noted daily. Pups were infected at day 25 after birth (4 days postweaning). Organs (brain, pancreas, and heart) were analyzed for viral RNA and histopathology. We observed that maternal infection at day 4 or day 17 of gestation had little effect on pregnancy outcome, whereas infection at day 10 affected dams and/or offspring. Infection of pups resulted in severe inflammation of the pancreas, but only when dams were previously infected, especially at day 17. The blood glucose levels were elevated. Because no trace of infection was found at the time of challenge, a role for immunopathology is suggested.

Introduction Enterovirus infections have usually a subclinical course, but they can cause severe diseases, particularly in neonates. These viruses frequently cause neonatal sepsis sometimes leading to disseminated intravascular coagulation, necrotizing hepatitis, and/or severe neurological and cardiac manifestations with a high mortality (Modlin, 1986; Galama, 1997). The frequency of neonatal enterovirus sepsis in the Netherlands is 10 times higher than that of neonatal herpes simplex infection, another condition with a potentially serious outcome (VerboonMaciolek et al., 2002; Poeran et al., 2008). The genus Enterovirus consists of 10 species of which seven are known human pathogens [Human enteroviruses (HEV) A, B, C, and D and Human rhinoviruses (HRV) A, B, and C]. Neonatal infections and chronic diseases as type 1 diabetes (T1D) and chronic myocarditis, where autoimmunity and/ or viral persistence may be involved, are associated with infection by viruses of the HEV-B genotype, which are the ª 2011 Federation of European Microbiological Societies Published by Blackwell Publishing Ltd. All rights reserved

most commonly diagnosed enteroviruses in clinical practice. Seroepidemiological surveys have associated enterovirus infection during pregnancy with increased risk for offspring to become diabetic, even years after birth (Dahlquist et al., 1995a, b; Hyo¨ty et al., 1995; Elfving et al., 2008). Few case reports suggest that infection during pregnancy may cause preterm delivery, fetal growth retardation, or even embryopathy (reviewed by Mata et al., 1977; Moore & Morens, 1984; Iwasaki et al., 1985; William et al., 1995; Keyserling, 1997; Cheng et al., 2006). However, these observations, which suggest that vertical transmission can take place, have still to be confirmed. So far, only a few experimental studies have been performed in mouse models on the influence of enterovirus infection during pregnancy (Dalldorf & Gifford, 1954; Soike, 1967; Modlin & Crumpacker, 1982). The objective of this study was to investigate the effect of maternal infection on pregnancy outcome and on infection of the offspring early in life. This was investigated in an outbred mouse model with virus inoculated by the oral route. FEMS Immunol Med Microbiol 64 (2012) 184–190

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Infection of pups of coxsackievirus B4 infected gravid mice

Materials and methods Virus strain and mice

For these studies, the coxsackievirus B4-E2 strain (CVB4E2), a diabetogenic strain was used. The strain was obtained with permission from J.W. Yoon (University of Calgary, Alberta, Canada). The virus was propagated in green monkey kidney cells. For the experiments, CD1 outbred male and female mice, aged 3–4 weeks, 15–17 g (Harlan Laboratories, Italy) were used. Infection of pregnant mice at different stages of pregnancy

For planned gestation, three females per male mouse were caged with sterile bedding, water, and mouse chow from Topdovo, Trnava, Slovak Republic. Successful fertilization was checked with vaginal swabs, to estimate the exact duration of gestation. Mice were infected at three different time points: days 4, 10, and 17 in the first, second, and third week of gestation, respectively. Two mice were infected per time point. For comparison, two mice per time point were mock-infected with PBS. Mice were infected with CVB4-E2 at a dose of 2 9 106 TCID50 by the oral route as described before (Bopegamage et al., 2005). Because of adverse outcome, infection at day 10 was repeated. Mice were weighed every day and observed for any signs of sickness. Loss in weight indicated severe fetal growth retardation, fetal death, and/or abortion. One dam, infected at day 10, became too sick to deliver and was euthanized near term. Infection of pups

Pups were separated from their mothers 3 weeks after birth (natural time for weaning) and put into separate cages, 3 per cage. To reduce effects of gender difference, only male pups were used. The pups were challenged orally 4 days after weaning (25 days after birth). The total number of pups per group was 6 : 3 infected pups and three controls. All pups (infected and mock-infected) were sacrificed and dissected at day 5 postinfection (p.i). Day 5 was chosen because preliminary experiments showed that at this time point the pancreas was affected and the glucose metabolism disturbed. Specimens collected

Mice were sacrificed after overnight fasting, and blood was drawn by cardiac puncture, performed by the direct visualization method (Hayward et al., 2007). Brain, heart, and pancreas were subsequently collected, partly snap-froFEMS Immunol Med Microbiol 64 (2012) 184–190

zen at 80 °C, and partly fixed in 4% formalin for histopathological analysis. Blood glucose levels were measured by means of a commercial system (Accu-Chek, Roche). Histology

The histological techniques and scoring of the grade (1– 4) of infiltration and necrosis were performed as described before (Bopegamage et al., 2005). Reverse transcriptase–polymerase chain reaction

Total RNA from the organs was extracted with PureLink RNA Mini kit (Invitrogen) according to the supplier’s manual for purifying total RNA from animal tissue. The details of the reverse transcription-PCR followed by nested PCR have been described previously (Bopegamage et al., 2005; de Leeuw 1994). For cDNA synthesis and amplification in a single tube, the SuperScript III One-Step RT-PCR System with Platinum Taq High Fidelity (Invitrogen) was used.

Results Effect of CVB4-E2 infection on the course of gestation

In all controls ( ), gestation was uneventful with a normal gain in weight (Fig. 1) and delivery of healthy litters of 14– 15 pups. [Correction added after online publication 6 December 2011: ( / ) changed to ( )]. After infection at days 4 and 17 of gestation, a normal course was observed with delivery of apparently healthy litters of 13–14 pups. Infection at day 10 (2nd week of gestation) showed an aberrant course (Fig. 1): Two of four dams aborted, and one showed a sudden loss of weight at 7 days p.i. After abortion, she remained healthy. The second dam lost activity and was lethargic. Her weight dropped also. She was euthanized: All fetuses were dead. The heart, pancreas, and brains of fetuses and of the dam were positive by PCR for viral RNA (not shown). The remaining two dams had litters of 6 and 10 pups, respectively. All 16 appeared healthy. Histology, fasting blood glucose levels, and presence of viral RNA in the organs of pups

All pups were sacrificed 5 days after challenge with virus or PBS. The mock-infected offspring ( / ) remained healthy; their organs were negative for viral RNA and organ tissue sections showed normal histology (Fig. 2a–c). Mock-infected offspring of dams infected at day 4, 10, or 17 of gestation, and a total of nine pups (+/ ) were also ª 2011 Federation of European Microbiological Societies Published by Blackwell Publishing Ltd. All rights reserved

S. Bopegamage et al.

186

70 60

Weight in gms

50

M1 Day 4 CVB4 E2

M3 Day 10 CVB4 E2

M2 Day 4 CVB4 E2

M4 Day 10 CVB4 E2

M1 Day 10 CVB4 E2

M1 Day 17 CVB4 E2

M2 Day 10 CVB4 E2

M2 Day 17 CVB4 E2

40 30 20 10 0

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 Gestation days

Fig. 1. Gain in weight of each CVB4-E2 infected dam. Arrows indicate the time of infection. M, dam; 1, 2, 3, number code of individuals per category.

(a)

(b)

(d)

(e)

(f)

(g)

negative by PCR and showed normal histology. Their blood glucose levels were in the normal range (Fig. 3). All virus-challenged offspring were PCR positive at day 5 p.i. in all tested organs. Major differences were observed, however, in histopathology (Fig. 2) and blood glucose values (Fig. 3), depending on whether or not the dam was previously infected (+/+ vs. /+) as well as on the day of maternal infection. Histological differences were prominent in the pancreas. Infected pups of mock-infected dams ( /+) showed only mild infiltration in the peripancreatic fat tissue (grade 1 of 4), but not in exocrine (acinar) or endocrine (islets) pancreatic tissues, which is in accord with our previous findings after infection by the oral route (Fig. 2d, Table 1) (Bopegamage et al., 2005). Brain and heart tissue of these pups were normal, as were blood glucose values (Table 1). In contrast, infected pups of dams that were infected at day 4 of gestation (+/+), displayed lymphocytic infiltrates

(c)

(h)

Fig. 2. Histology of organs of challenged offspring of CVB4-E2 infected and control dams. (a) Control brain, offspring / *, mother mock infected day 4, (40 9). (b) Control pancreas, offspring / , mother mock infected day 4, (20 9). (c) Control heart, offspring / , mother mock infected day 4, (40 9). (d) Infiltration in the peripancreatic fat tissue as seen in, offspring /+, mothers mock infected on day 4 or 10 or 17, (40 9). (e) Interstitial infiltrates in the pancreatic tissues, offspring +/+, mother infected on day 4, (40 9). (f ) Oedema, capillary hyperemia in the brain, offspring +/+, mother infected on day 17, (40 9). (g) Acute pancreatitis with focal necrosis of pancreatic acines, dense mixed inflammatory infiltrates, offspring +/+, mother infected on day 17, (20 9). (h) Heart focal intersticial infiltrate with lymphocytes, no dystrophic change of myocytes in offspring +/+, mother infected on day 17, (40 9). *+, infection with CB4-E2; , mock infection with PBS; +/ , mother infected and offspring mock infected; /+, mother mock infected and offspring infected; +/+, mother and offspring infected; / , mother and offspring mock infected.

ª 2011 Federation of European Microbiological Societies Published by Blackwell Publishing Ltd. All rights reserved

FEMS Immunol Med Microbiol 64 (2012) 184–190

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Infection of pups of coxsackievirus B4 infected gravid mice

22 Pup 1

20

Pup 2 Pup 3

18

Glucose mmol L–1

16 14 12 10 8 6 4 2 0

+/+*

+/–

–/+

–/–

Offspring of dams infected at day 4

+/+

+/–

–/+

–/–

Offspring of dams infected at day 10

+/+

+/–

–/+

–/–

Offspring of dams infected at day 17

Fig. 3. Fasting blood glucose values of the challenged offspring of CVB4 E2 infected and control dams. *+, infection with CB4-E2; , mock infection with PBS; +/ , mother infected and offspring mock infected; /+, mother mock infected and offspring infected; +/+, mother and offspring infected; / , mother and offspring mock infected.

Table 1. Infiltration scores in the pancreas P1 Offspring category Day 4 Grade of infiltration Acinar tissue Peripancreatic fat tissue Day 10 Grade of infiltration Acinar tissue Peripancreatic fat tissue Day 17 Grade of infiltration Acinar tissue Peripancreatic fat tissue

P2

P3

(+/+)*

P1

P2

P3

(+/ )

P1

P2

P3

( /+)

P1

P2

P3

( / )

3 2

3 2

2 3

0 0

0 0

0 0

0 1

0 1

0 1

0 0

0 0

0 0

0 1

0 1

0 2

0 0

0 0

0 0

0 1

0 1

0 1

0 0

0 0

0 0

4 2

4 2

4 2

0 0

0 0

0 0

0 1

0 1

0 1

0 0

0 0

0 0

P, pup; 1, 2, 3, number code of individual per category; *+, infection with CB4-E2; , mock infection with PBS; +/ , mother infected and offspring mock-infected; /+, mother mock-infected and offspring infected; +/+, mother and offspring infected; / , mother and offspring mockinfected.

(grade 2–3), not only in the peripancreatic fat tissue but also in acinar tissue of the pancreas (Fig. 2e, Table 1). Islets appeared microscopically unaffected, but the glucose values were clearly elevated (16.7–19.7 mM). Infected pups of dams infected at day 10 had little infiltration in the peripancreatic fat tissue (grade 1). The acinar tissue was unaffected as were the islets, and only one mouse had a slightly elevated glucose value of 11.4 mM (Table 1) as compared to the controls. Infected pups of dams infected at day 17 showed dense lymphocytic infiltrates with severe necrosis (grade 4) in acinar tissue (Fig. 2g) and infiltration in the peripancreatFEMS Immunol Med Microbiol 64 (2012) 184–190

ic fat tissue (grade 2). Again, no infiltrates were seen in the islets, but blood glucose values were mildly elevated (11.0–15.4 mM). In contrast to offspring of dams, infected at days 4 and 10, these pups showed edema and capillary hyperemia in their brains without infiltrates (Fig. 2f ) but showed minor changes with sparse focal infiltrates in their hearts (Fig. 2h).

Discussion Our study revealed several remarkable outcomes: (1) Infection in the second week of gestation was harmful for ª 2011 Federation of European Microbiological Societies Published by Blackwell Publishing Ltd. All rights reserved

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dams and subsequently for outcome of gestation (stillbirth, abortion, and reduced litter seize); (2) Infection during gestation influenced the severity of postnatal infection in pups upon homologous challenge; and (3) Upon challenge, the histopathology and the function of the pancreas were mostly affected. Spontaneous abortion and sickness of the mother after infection in the second week are comparable to findings of Modlin & Crumpacker (1982). They explained spontaneous abortions by vertical transmission of virus. These authors and others (Dalldorf & Gifford, 1954) attributed the difference in susceptibility to infection of dams during the second and third weeks (days 10 and 17 in our study) to physiological changes in hormone levels, which are associated with diminished immunity. All nine control pups of infected dams (+/ ) were negative by PCR at day 30 after birth and showed normal histology. In our mouse model, CVB infections were not cleared within 30 days (Bopegamage et al., 2005). Therefore, the negative PCR results in these (+/ ) pups do suggest that transplacental infection did not occur, but period shortly after birth needs to be investigated to confirm this observation. Upon homologous challenge (+/+), the infection was clearly more severe than it was in offspring of control dams ( /+). It was most severe in offspring of dams infected in the third week (day 17) of gestation, affecting brain, heart, and pancreas. Necrosis and infiltration of the pancreatic tissue were massive and more severe than the histopathological changes observed in pups of dams infected at day 4 or 10 of gestation. The extensive pathology in the pancreas, as compared to heart and brain tissues, which was found in all three groups, could be due to the diabetogenic properties of the virus strain. It would be interesting to repeat the study with nonadapted wild strains. The difference in fasting glucose levels between offspring of dams infected at days 4 and 17, compared with the offspring of dams infected at day 10, is statistically significant (Student’s t-test: P < 0.05). The underlying mechanisms and the reason why the glucose levels were not increased in all challenged pups is not well understood. Insulin staining of islets and virus titration of the pancreases did not reveal significant differences and, therefore, could not account for the variation in glucose metabolism (data not shown). Further investigation is required in this area, and we can only speculate about the cause. It may be related to the developmental stage of the embryo and of its immune system at the time of infection of the mother. The relatively mild pathology in the surviving challenged offspring of the dams infected at day 10 upon is also difficult to understand and requires further investigation. ª 2011 Federation of European Microbiological Societies Published by Blackwell Publishing Ltd. All rights reserved

S. Bopegamage et al.

Several studies reported enhanced pathology after a heterologous challenge of adult mice with CVB3 after an initial infection with CVB2 (Beck et al., 1990; Yu et al., 1999; Michels & Tiu, 2007). In these studies, a heterologous challenge was crucial for enhanced pathology, suggesting an effect of cross-reactivity and enhanced immunopathology which may be due to the phenomenon of original antigenic sin (Morens et al., 2010) or to antibody-dependent enhancement (ADE) (Beck et al., 1990; Girn et al., 2002; Kishimoto et al., 2002; Takada & Kawaoka, 2003; Sauter & Hober, 2009). Our data have more similarity to those of Horwitz et al. (2003), who showed that in adult mice homologous challenge with CVB4-E2 resulted in hyperglycemia. The authors showed that the effect was not directly T-cellmediated although T cells were still essential for survival of infection. We hypothesize on the basis of our data that preexisting immunity is responsible for the enhanced pathology in the offspring and that the observed effects are thus immune-mediated. There are several options: (1) maternal antibodies, passively transferred to offspring; (2) T-cell-mediated immunity, and (3) triggering of autoimmunity. Implications of these options are the following: (1) maternal antibodies are expected to be of the neutralizing type being able to protect pups from infection with the homologous strain; however, low antibody levels may fail to neutralize the virus and cause an adverse effect by means of ADE as has been reported before (Beck et al., 1990; Girn et al., 2002; Horwitz et al., 2003; Takada & Kawaoka, 2003; Sauter & Hober, 2009). Indeed, antibodies were present in the 9 (+/ ) control pups and in the infected dams. Assuming that the offspring were not infected antenatally as we believe, the antibodies must have been of maternal origin; (2) intrauterine infection of the pups may raise a cellular immune response which, because of a gradual maturation of the fetal immune system, may be more vigorous in the 3rd week of gestation than in earlier stages. The latter can explain the more severe course upon challenge after maternal infection at day 17; (3) autoimmunity, being actually a variant of option (2), may be triggered by infection of pancreatic islets of the mother, thus presenting islet auto-antigens in a context of (infectious) danger signaling during the development of the fetus. For the latter two options, an antenatal infection may probably not be needed, as recently was shown by Jubayer et al. (2010), who demonstrated that postnatal immunity can be specifically raised by immunization of the mother during gestation. Hence, all three mechanisms (passive transfer of antibody and induction of cellular immunity against viral and/or auto-antigens) may thus occur in the absence of antenatal infection. Further studies are FEMS Immunol Med Microbiol 64 (2012) 184–190

Infection of pups of coxsackievirus B4 infected gravid mice

required to investigate which of these possibilities are responsible for the enhanced pathology. An important implication of this study is that maternal infections during pregnancy can play a role in the course of subsequent infections in offspring, which may have chronic consequences, such as the triggering of T1D. Several serological studies from Sweden (Dahlquist et al., 1995a, b) and Finland (Hyo¨ty et al., 1995; Elfving et al., 2008) support a relationship between maternal enterovirus infection during pregnancy and T1D in the offspring, established by the age of 10 years or even later. However, enterovirus infections in the 1st trimester were not a risk factor (Viskari et al., 2002), which is not in accordance with the outcome of our experimental study. The present study shows for the first time that infection of outbred mice by oral route during gestation results in enhanced pathology upon postnatal challenge of the offspring with the homologous virus strain. The pathology was mainly confined to the pancreas and resulted in hyperglycemia. This observation provides a new model for study of the still enigmatic cause of T1D.

Acknowledgements The funding is provided by the Norwegian financial support mechanism, Mechanism EEA, and Slovak Government – Project SK0082 and received by S.B. The authors declare no conflict of interest. We thank Bill Coleman, Texas, USA, for proof reading and suggestions.

Ethical approval Permission for the animal work was obtained from the Ethics Committee of the Slovak Health University and the State Veterinary and Food Control Authority of the Slovak Republic.

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