Enteroviruses (family: Picornaviridae) and especially
Coxsackievirus B (CVB) have often been implicated in
the etiology of type 1 diabetes mellitus. Enteroviral
RNA has been found, by several investigators, in
peripheral blood of 27 to 64% of patients with type 1
diabetes (12). In some cases, CVB RNA sequences
showed a significant homology with CVB4 (6). In
another study, sequences of CVB4 RNA homologous
to coxsackievirus B4 E2 (CVB4 E2) were found in
peripheral blood mononuclear cells of patients with
type 1 diabetes (20). CVB4 E2 is the best known dia-
betogenic enterovirus strain. It was isolated from the
pancreas of a child who died from diabetic ketoacidosis
and it was able to destroy β cells of the pancreas and to
induce hyperglycemia in some susceptible mouse
strains (21). A persistent infection of human pancreatic
islet cells and thymic epithelial cells in vitro can be
obtained with CVB4 E2 (3, 5).
The spreading of CVB4 E2 following its introduc-
tion into the organism through a natural way has not
been studied yet. Therefore, we developed an experi-
mental model based on an oral inoculation of outbred
Swiss Albino mice with CVB4 E2. The aim of the cur-
rent study was to determine the targets of the infection
and the kinetics of viral RNA in various tissues, particu-
larly in lymphoid organs and blood.
Briefly, 3–4 weeks old Swiss Albino female mice
(Pasteur Institute, Tunis, Tunisia), were orally inoculat-
ed, by using a rigid canula, with 104.74TCID50of CVB4
E2 contained in 200 µl culture supernatant of infected-
cells. CVB4 E2 (kindly provided by Dr. J.W. Yoon,
Calgary, Canada) was propagated in Hep-2 cells
(BioWhittaker) in Eagle’s minimal essential medium
(Gibco BRL) supplemented with 10% decomplemented
fetal calf serum and 1% L-glutamine. Mice were treated
according to general ethical rules and maintained under
specific pathogen-free conditions with unlimited access
to food and water. Two infected animals were sacri-
ficed at different post-infectious (p.i.) times ranging
from 1 hr to 180 days. Naïve mice served as negative
Microbiol. Immunol., 50(12), 971–974, 2006
*Address correspondence to Dr. Didier Hober, Service de
Virologie/UPRES EA3610, Faculté de Médecine, Université
Lille 2, Bâtiment Paul Boulanger, CHRU Lille, 59037 Lille,
France. Fax: ?33–3–20–44–52–81. E-mail: dhober@chru-
Abbreviations: CVB4 E2, coxsackievirus B4 E2; p.i., post-
infectious; sn-RT-PCR, semi-nested-RT-PCR.
Prolonged Viral RNA Detection in Blood and
Lymphoid Tissues from Coxsackievirus B4 E2
Orally-Inoculated Swiss Mice
Hela Jaïdane1, 2, Jawhar Gharbi2, Pierre-Emmanuel Lobert1, Bernadette Lucas1, Raïda Hiar2,
Manel Ben M’Hadheb2, Fabienne Brilot3, Vincent Geenen3, Mahjoub Aouni2, and
Didier Hober*, 1
1Service de Virologie/UPRES EA3610, Faculté de Médecine, Université Lille 2, Bâtiment Paul Boulanger, CHRU Lille,
59037 Lille, France, 2Laboratoire Lab-MDT-01, Unité de Pathogenèse & Virulence Virales, Faculté de Pharmacie de Mona-
stir, Avenue Avicenne 5000 Monastir, Tunisia, and 3Université de Liège, Centre d’Immunologie de Liège, Institut de Pathologie
4, CHU B-23, B-4000 Liège (Sart-Tilman), Belgium
Received July 31, 2006; in revised form, August 24, 2006. Accepted September 2, 2006
Abstract: The spreading of viral RNA within Swiss Albino mice orally inoculated with coxsackievirus B4 E2
strain (CVB4 E2) was studied by using RT-PCR and semi-nested-RT-PCR methods. Viral RNA was
detected in various organs: pancreas, heart, small intestine, spleen, thymus, and blood at various post-
infectious (p.i.) times ranging from 8 hr to 150 days. Our results show that (i) outbred mice can be infected
with CVB4 E2 following an oral inoculation, which results in systemic spreading of viral RNA, (ii) CVB4
E2 infection can be associated with a prolonged detection of viral RNA in spleen, thymus and blood, up to
70 days p.i. and further in other organ tissues.
Key words: Coxsackievirus B4, Mouse, RT-PCR, Thymus
controls. From each animal, blood was collected on
EDTA and heart, thymus, pancreas, spleen, small intes-
tine were removed, rinsed with PBS, snap-frozen in liq-
uid nitrogen and stored at ?80 C. Total RNA was
extracted using the RNAgents Total RNA Isolation
System kit (Promega) for blood samples, and using Tri-
Reagent (Sigma) for other tissues, as described by
Chomczynski and Sacchi, (7). Genome amplification
was then performed using a single-tube method with
the SuperScriptTMOne-Step RT-PCR with Platinum®
Taq kit (Invitrogen), as previously described (10).
Primer sense 006: 5'-TCCTCCGGCCCCTGAATGCG-
3' and anti-sense 007: 5'-ATTGTCACCATAAGCAGC-
CA-3' (Proligo) were selected within the 5' non-translat-
ed region of the enteroviral genome; generating a 155
bp fragment (22). Total RNA extracts from samples
showing negative results by this method were submitted
to a RT-PCR similar to the previous one except that the
sense-primer 006 was replaced by an external one that
we called 008 5'-GAGTATCAATAAGCTGCTTG-3'
(Proligo) also located within the 5' non-translated
region, generating a 414 bp fragment. A semi-nested-
PCR was then performed on first amplification prod-
ucts, using 006 and 007 primers and the Platinum PCR
SuperMix kit (Invitrogen) as recommended by the man-
ufacturers. For each RNA sample, GAPDH mRNA
was submitted to RT-PCR, using primer sense 5'-AAC-
GACCCCTTCATTGAC-3' and anti-sense 5'-TCCAC-
GACATACTCAGCAC-3' (Proligo), and used as a posi-
tive control to prove the absence of reaction inhibitors.
The amplified products were analyzed by electrophore-
sis on a 2% agarose gel containing 0.5 mg ml?1of
ethidium bromide (Sigma) and visualized by using the
Gel Doc 2000 system (Bio-Rad).
This is the first study describing the detection of viral
RNA in CVB4 E2 orally-infected outbred mice. Table 1
gives all RT-PCR results and those of semi-nested-RT-
PCR (sn-RT-PCR) that were positive. For control mice,
all sampled tissues were negative (data not shown). For
inoculated animals, CVB4 E2 RNA was found in all
sampled tissue types at various p.i. times ranging from 8
hr to 150 days: from 16 hr up to 70 days in blood, thy-
mus and heart, from 16 hr up to 90 days in pancreas,
from 8 hr up to 70 days in spleen, and from 8 hr up to
150 days in small intestine. All samples, including
those showing negative results for CVB4 E2 RNA,
were positive for GAPDH mRNA, proving the RNA
integrity and the absence of reaction inhibitors (results
not shown). Our data show that a systemic spreading of
CVB4 E2 following oral inoculation is possible and
that outbred mice, in addition to inbred, diabetic,
immuno-compromised or transgenic mice (4, 8, 9, 11,
18, 21), can be infected with that viral strain. The nat-
ural expression in the mouse of a coxsackievirus and
adenovirus receptor strongly homologous to the human
one (19), made possible the use of these animals as
models to study the infection with CVB4 E2. We
decided to use per-oral infected outbred mice in our
experiments because such animals are much more rep-
resentative of the natural variation in human population
than inbred mice as underlined by other authors (1) and
because it is important to consider the natural route of
infection in experimental models.
CVB4 E2 strain was capable of targeting several
organs, and its RNA was detectable up to 70 days and
further (see Table 1). Our results concerning a pro-
longed viral RNA detection in heart, spleen, pancreas
and small intestine are in agreement with those of other
H. JA¨IDANE ET AL
Table 1. RT-PCR (and sn-RT-PCR) results on sampled tissues from CVB4 E2 orally-infected Swiss mice
Time p. i.
p.i.: post-infection; hr: hour; d: day; NS: not sampled.
Two mice were tested at each time point (??: two mice showing positive results by RT-PCR; ??: two mice showing negative results by RT-
PCR; ??: one mouse showing positive and another one showing negative result by RT-PCR).
sn-RT-PCR was performed only for samples showing negative results by RT-PCR. Only positive results by sn-RT-PCR are mentioned (sn?: one
positive result by sn-RT-PCR, 2sn?: two positive results by sn-RT-PCR).
authors obtained in mice inoculated with CVB3 Nancy
by the oral route (2, 10). The prolonged detection of
viral RNA in the spleen is reminiscent of results of
other investigations based on intraperitoneal inoculation
by CVB3 Nancy (14, 16). The detection of viral RNA
by sn-RT-PCR in pancreatic tissue was possible up to 90
days p.i. in our experiments with CVB4 E2 and up to 56
days p.i. in another study with CVB3 (2), which can be
due to a stronger pancreatotropic property of CVB4 E2.
Indeed, it has been reported that the pancreases of CD-1
mice intraperitoneally challenged with CVB4 E2, were
still positive for viral RNA at 6 months p.i. (17).
Our results show that CVB4 E2 RNA can reach the
thymus in vivo following an infection by a natural route
and that, in these conditions, viral RNA can be found in
thymus until 70 days p.i. (like in spleen and heart). In a
previous investigation based on CVB3 intraperitoneal
inoculation of SWR/J, H-2q mice, viral RNA in thymus
was visualized by in situ hybridization, at a low level at
day 6 but not at day 42 p.i. (14). In mice inoculated
with CVB4 E2 through the intraperitoneal route, Chat-
terjee et al. (4) obtained a thymic infection; the virus
was rapidly cleared from that organ but at 8 weeks p.i. a
150% increase in CD4?CD8?thymic T cells was
observed. It has been reported that a viral infection of
thymus can facilitate the immune tolerance to antigens
of that infectious agent, which can result in viral persis-
tence (13, 15). In the human system, a prolonged
detection of CVB4 E2 RNA has been obtained in
thymic cells infected in vitro, which resulted in a sus-
tained production of cytokines by these cells (3). In so
far as CVB4 E2 RNA can be detected in thymus in
experimental models, particularly after oral infection as
demonstrated here, on one hand, and a CVB4 can dis-
turb the function of that organ on the other hand, further
studies are required to determine whether CVB4 infec-
tions in humans can modulate the thymic function and
whether that can play a role in CVB4-induced diseases.
To the best of our knowledge, this is the first investi-
gation reporting a prolonged detection of CVB RNA in
mice blood. Our interest in the blood comes from the
fact that enteroviral RNA of CVB subgroup members,
especially CVB4, was detected in human blood from
patients with type 1 diabetes with nucleotide sequence
homologous to CVB4 E2 in some patients (6, 20). In an
experimental model based on inbred male SWR mice
inoculated with CVB3 by the intraperitoneal route (16),
viral RNA was detected by quantitative RT-PCR in
blood up to day 14 p.i. In the current study, CVB4 E2
RNA was detected from 16 hr up to 70 days p.i. in
blood (see Table 1). However, CVB4 E2 RNA was not
detected in every sample of blood or other tissues (see
Table 1). For example, CVB4 E2 RNA was detected in
pancreatic samples at different p.i. times, from 16 hr up
to 90 days, but neither at 42 days nor at 70 days. The
discordance of viral RNA detection in blood and organs
suggest that the positive RT-PCR tests for organs were
not due to a contamination with viral RNA-containing
blood and argue in favor of the presence of viral
nucleotide sequences in the studied organ. Together,
the variations of the detection of CVB4 E2 RNA in
blood and other tissues in our experiments suggest that
the circulation of enteroviral RNA within the organism
can be different at various p.i. times. An alternative
explanation is that our data reflect a different response to
the infection due to natural individual variations within
In our experiments, viral RNA was detected as soon
as 8 hr p.i. in spleen and small intestine, and 16 hr p.i. in
the other tissue types. In the investigation conducted by
Harrath et al. (10), per-oral infection of BALB/c mice
with CVB3 Nancy affected the different organs in a
more progressive way (the intestine at 2 hr, the heart at
1 day, the pancreas at 2 days and the spleen at 3 days
p.i.). That difference can be attributed to distinct viral
and mice strains. Our data suggest that CVB4 E2 can
rapidly pass through the gut to reach the spleen and
then spread towards various organs.
Our experimental model of CVB4 E2 infection by a
natural route shows that viral RNA can be found by RT-
PCR in blood and in immune system organs, spleen and
thymus, up to 70 days p.i. In addition to these organs,
CVB4 E2 RNA was detected in small intestine, heart
and pancreas up to 56 days by RT-PCR and further by
sn-RT-PCR. Together our data suggest that CVB4 E2
infection by a natural route can be associated with a
prolonged detection of viral RNA within the organism,
and that viral RNA can be detected in blood. Studies
are in progress in our laboratory to determine whether
the detection of enteroviral RNA in peripheral blood of
patients with type 1 diabetes is related to a prolonged
presence of enteroviruses which would be consistent
with a persistent infection with these viruses in such
We thank Rafik Harrath and Houda Daami, from Laboratoire
de Virologie, Faculté de Pharmacie, Monastir, Tunisia, and Del-
phine Caloone and Cécile Schanen from UPRES EA3610, Uni-
versité Lille 2, France, for helpful assistance.
Hela Jaïdane was supported by the Comité Mixte de
Coopération Universitaire (CMCU) with grants from Egide
Paris. This work was supported by Ministère de la Recherche
Scientifique, de la Technologie et du développement des
compétences (Lab-MDT-01), Tunisia, Ministère de l’Education
Nationale de la Recherche et de la Technologie, Université Lille
2 (UPRES EA3610), France, and by the EU FP6 Integrated Pro-
ject EURO-THYMAIDE (Contract LSHB-CT-2003-503410).
1) Bopegamage, S., Borsanyiova, M., Vargova, A., Petrovico-
va, A., Benkovicova, M., and Gomolcak, P. 2003. Coxsack-
ievirus infection of mice. I. Viral kinetics and histopatho-
logical changes in mice experimentally infected with cox-
sackieviruses B3 and B4 by oral route. Acta Virol. 47:
2) Bopegamage, S., Kovacova, J., Vargova, A., Motusova, J.,
Petrovicova, A., Benkovicova, M., Gomolcak, P., Bakkers,
J., van Kuppeveld, F., Melchers, W.J.G., and Galama, J.M.
2005. Coxsackie B virus infection of mice: inoculation by
the oral route protects the pancreas from damage, but not
from infection. J. Gen. Virol. 86: 3271–3280.
3) Brilot, F., Chehadeh, W., Charlet-Renard, C., Martens, H.,
Geenen, V., and Hober, D. 2002. Persistent infection of
human thymic epithelial cells by coxsackievirus B4. J.
Virol. 76: 5260–5265.
4) Chatterjee, N.K., Hou, J., Dockstader, P., and Charbonneau,
T. 1992. Coxsackievirus B4 infection alters thymic, splenic,
and peripheral lymphocyte repertoire preceding onset of
hyperglycemia in mice. J. Med. Virol. 38: 124–131.
5) Chehadeh, W., Kerr-Conte, J., Pattou, F., Alm, G., Lefebvre,
J., Wattre, P., and Hober, D. 2000. Persistent infection of
human pancreatic islets by coxsackievirus B is associated
with alpha interferon synthesis in beta cells. J. Virol. 74:
6) Chehadeh, W., Weill, J., Vantyghem, M.C., Alm, G., Lefeb-
vre, J., Wattre, P., and Hober, D. 2000. Increased level of
interferon-α in blood of patients with insulin-dependent
diabetes mellitus: relationship with coxsackievirus B infec-
tion. J. Infect. Dis. 181: 1929–1939.
7) Chomczynski, P., and Sacchi, N. 1987. Single-step method
of RNA isolation by acid guanidinium thiocyanate-phenol-
chloroform extraction. Anal. Biochem. 162: 156–159.
8) Cook, S.H., Loria, R.M., and Madge, G.E. 1982. Host fac-
tors in Coxsackievirus B4-induced pancreopathy. Lab.
Invest. 46: 377–382.
9) Gerling, I., and Chatterjee, N.K. 1993. Lack of 64,000-M(r)
islet autoantigen overexpression and antibody development
following coxsackievirus B4 infection in diabetes-resistant
mice. Autoimmunity 14: 197–203.
10) Harrath, R., Douche-Aourik, F., Bourlet, T., Ismail, A.,
Omar, S., Aouni, M., and Pozzetto, B. 2004. Model of cox-
sackievirus B3 persistent infection in orally-inoculated
BALB/c mouse. Ann. Biol. Clin. 62: 33–39.
11) Horwitz, M.S., Ilic, A., Fine, C., Rodriguez, E., and Sarvet-
nick, N. 2002. Presented antigen from damaged pancreatic
beta cells activates autoreactive T cells in virus-mediated
autoimmune diabetes. J. Clin. Invest. 109: 79–87.
12) Hyôty, H. 2002. Enterovirus infections and type 1 diabetes.
Ann. Med. 34: 138–147.
13) King, C.C., Jamieson, B.D., Reddy, K., Bali, N., Concep-
cion, R.J., and Ahmed, R. 1992. Viral infection of the thy-
mus. J. Virol. 66: 3155–3160.
14) Klingel, K., Stephan, S., Sauter, M., Zell, R., McManus,
B.M., Bültmann, B., and Kandolf, R. 1996. Pathogenesis of
murine enterovirus myocarditis: virus dissemination and
immune cell targets. J. Virol. 70: 8888–8895.
15) Korostoff, J.M., Nakada, M.T., Faas, S.J., Blank, K.J., and
Gaulton, G.N. 1990. Neonatal exposure to thymotropic
gross murine leukemia virus induces virus-specific
immunologic nonresponsiveness. J. Exp. Med. 172: 1765–
16) Reetoo, K.N., Osman, S.A., Illavia, S.J., Cameron-Wilson,
C.L., Banatvala, J.E., and Muir, P. 2000. Quantitative
analysis of viral RNA kinetics in coxsackievirus B3-
induced murine myocarditis: biphasic pattern of clearance
following acute infection, with persistence of residual viral
RNA throughout and beyond the inflammatory phase of
disease. J. Gen. Virol. 81: 2755–2762.
17) See, D.M., and Tilles, J.G. 1995. Pathogenesis of virus-
induced diabetes in mice. J. Infect. Dis. 171: 1131–1138.
18) Serreze, D.V., Wasserfall, C., Ottendorfer, E.W., Stalvey,
M., Pierce, M.A., Gauntt, C., O’Donnell, B., Flanagan,
J.B., Campbell-Thompson, M., Ellis, T.M., and Atkinson,
M.A. 2005. Diabetes acceleration or prevention by a cox-
sackievirus B4 infection: critical requirements for both
interleukin-4 and gamma interferon. J. Virol. 79:
19) Tomko, R.P., Xu, R., and Philipson, L. 1997. HCAR and
MCAR: the human and mouse cellular receptors for sub-
group C adenoviruses and group B coxsackieviruses. Proc.
Natl. Acad. Sci. U.S.A. 94: 3352–3356.
20) Yin, H., Berg, A.K., Tuvemo, T., and Frisk, G. 2002.
Enterovirus RNA is found in peripheral blood mononuclear
cells in a majority of type 1 diabetic children at onset. Dia-
betes 51: 1964–1971.
21) Yoon, J.W., Austin, M., Onodera, T., and Notkins, A.L.
1979. Isolation of a virus from the pancreas of a child with
diabetic ketoacidosis (virus-induced diabetes mellitus). N.
Engl. J. Med. 300: 1173–1179.
22) Zoll, G.J., Melchers, W.J., Kopecka, H., Jambroes, G., van
der Poel, H.J., and Galama, J.M. 1992. General primer-
mediated polymerase chain reaction for detection of
enteroviruses: application for diagnostic routine and persis-
tent infections. J. Clin. Microbiol. 30: 160–165.
H. JA¨IDANE ET AL