JOURNAL OF VIROLOGY, July 2008, p. 6139–6149
Copyright © 2008, American Society for Microbiology. All Rights Reserved.
Vol. 82, No. 13
Rotavirus Infection Accelerates Type 1 Diabetes in Mice with
Kate L. Graham,1†‡ Natalie Sanders,1†‡ Yan Tan,1§ Janette Allison,1,2
Thomas W. H. Kay,2and Barbara S. Coulson1*
Department of Microbiology and Immunology, The University of Melbourne, Victoria 3010, Australia,1and
St. Vincent’s Institute, Fitzroy, Victoria 3065, Australia2
Received 18 March 2008/Accepted 9 April 2008
Infection modulates type 1 diabetes, a common autoimmune disease characterized by the destruction of
insulin-producing islet ? cells in the pancreas. Childhood rotavirus infections have been associated with
exacerbations in islet autoimmunity. Nonobese diabetic (NOD) mice develop lymphocytic islet infiltration
(insulitis) and then clinical diabetes, whereas NOD8.3 TCR mice, transgenic for a T-cell receptor (TCR)
specific for an important islet autoantigen, show more rapid diabetes onset. Oral infection of infant NOD
mice with the monkey rotavirus strain RRV delays diabetes development. Here, the effect of RRV infection
on diabetes development once insulitis is established was determined. NOD and NOD8.3 TCR mice were
inoculated with RRV aged >12 and 5 weeks, respectively. Diabetes onset was significantly accelerated in
both models (P < 0.024), although RRV infection was asymptomatic and confined to the intestine. The
degree of diabetes acceleration was related to the serum antibody titer to RRV. RRV-infected NOD mice
showed a possible trend toward increased insulitis development. Infected males showed increased CD8?
T-cell proportions in islets. Levels of ?-cell major histocompatibility complex class I expression and islet
tumor necrosis factor alpha mRNA were elevated in at least one model. NOD mouse exposure to mouse
rotavirus in a natural experiment also accelerated diabetes. Thus, rotavirus infection after ?-cell auto-
immunity is established affects insulitis and exacerbates diabetes. A possible mechanism involves in-
creased exposure of ? cells to immune recognition and activation of autoreactive T cells by proinflam-
matory cytokines. The timing of infection relative to mouse age and degree of insulitis determines whether
diabetes onset is delayed, unaltered, or accelerated.
Type 1 diabetes results from an autoimmune process in
which pancreatic ? cells are selectively destroyed. An islet
lymphoid infiltrate develops that is described as “insulitis”
(49). Virus infections are proposed to play a role in type 1
diabetes development through ?-cell cytolysis or loss of self-
tolerance following pancreatic infection, bystander activation
of T cells, and molecular mimicry between ? cell autoantigens
and viral epitopes (19, 50, 53).
Rotaviruses are the major agents of severe acute gastroen-
teritis in children and have been implicated in exacerbation of
type 1 diabetes development (26). Antibody seroconversion to
rotavirus in Australian children was associated with increases
in autoantibodies to glutamic acid decarboxylase (GAD) and
insuloma-associated protein 2 tyrosine phosphatase (IA-2).
Amino acid sequence similarity between rotavirus protein VP7
and T-cell epitopes in human GAD and IA-2 led to the sug-
gestion of T-cell molecular mimicry as a possible mechanism
(26, 27). Although later studies in Finnish children did not
confirm the association between rotavirus infection and islet
autoimmunity (6, 34), increased antibody responses to dietary
bovine insulin were noted after rotavirus infection (35). Addi-
tional findings that might support links between rotavirus in-
fection and other autoimmunity-related diseases also have
been reported (33, 47, 57, 58).
The nonobese diabetic (NOD) mouse spontaneously devel-
ops a form of autoimmune diabetes similar to human type 1
diabetes (3, 46). Most mice show severe insulitis by 10 weeks of
age. By 30 weeks of age the diabetes incidence typically reaches
60 to 80% in NOD females and 10 to 20% in NOD males.
NOD diabetes mainly depends on CD4?and CD8?T cells,
and most cells in the insulitic lesion are CD4?T cells. Auto-
reactive T cells are primed in the draining pancreatic lymph
node(s) (PLN) and then migrate to the islets (20, 24, 29). Like
humans, NOD mice produce autoantibodies and T cells to
GAD and insulin. In addition, CD8?T cells directed to the
islet-specific glucose 6-phosphatase catalytic subunit-related
protein (IGRP) are an important component of islet-infiltrat-
ing T cells in prediabetic NOD mice (2, 15, 31, 41). Circulating
IGRP-reactive T-cell numbers predict diabetes in NOD mice
and new-onset patients (36, 52). Expression of the IGRP-
specific T-cell receptor (TCR) in NOD mice (NOD8.3 TCR)
led to 8.3 TCR expression on ?90% of islet-infiltrating T cells
and a high diabetes incidence with rapid onset (54, 55).
NOD8.3 TCR mice provide a simplified and rapid mouse
model of spontaneous diabetes and a useful tool to study the
role of CD8?T cells.
Murine rotaviruses and the rhesus monkey rotavirus strain
RRV induce diarrhea in infant mice and infect intestinal cells
* Corresponding author. Mailing address: Department of Microbi-
ology and Immunology, Gate 11, Royal Parade, The University of
Melbourne, Melbourne, Victoria 3010, Australia. Phone: 61 3 8344
8823. Fax: 61 3 9347 1540. E-mail: email@example.com.
† K.L.G. and N.S. contributed equally to this study.
‡ Present address: St. Vincent’s Institute, Fitzroy, Victoria 3065,
§ Present address: Department of Dentistry, The University of Mel-
bourne, Victoria 3010, Australia.
?Published ahead of print on 16 April 2008.
without causing disease in adults, although the doses required
differ by several logs (8, 38, 56). Oral RRV infection of infant
NOD mice causes gastroenteritis and delays diabetes onset,
whereas infection in young adult NOD mice without estab-
lished insulitis is asymptomatic and diabetes is unaffected (21).
RRV infection in infant or young adult NOD mice does not
initiate insulitis (21). Infectious RRV spreads to the pancreas
in infant NOD mice, with viral antigen localized in macro-
phages outside islets. Although RRV replicates in islets and
pancreatic cells isolated from young adult NOD mice, infec-
tious virus is not detectable in the pancreases of orally inocu-
lated mice of this age (11, 21).
The effect of RRV rotavirus infection on insulitis and dia-
betes development in older prediabetic NOD and NOD8.3
TCR mice with established insulitis was examined in the
present study. RRV is shown here to accelerate diabetes onset
and incidence in older prediabetic mice in the absence of
detectable extraintestinal spread and pancreatic infection.
NOD mice exposed to mouse rotavirus in a natural experiment
also showed increased diabetes development. These data pro-
vide the first evidence that rotavirus infection can accelerate
diabetes development in an animal model.
MATERIALS AND METHODS
Mice. NOD and BALB/c mice were obtained from the Animal Resources
Centre (Canning Vale, Western Australia, Australia). NOD8.3 TCR mice, ex-
pressing the TCR?? rearrangements of the H-2Kd-restricted, islet ?-cell-reactive
CD8?T-cell clone NY8.3 on a NOD genetic background (54), were provided by
P. Santamaria (University of Calgary, Calgary, Alberta, Canada). Mice were bred
and housed in the animal facility of the Department of Microbiology and Im-
munology at the University of Melbourne in isolators under specific-pathogen-
free conditions as described previously (21). Offspring of transgenic mice were
screened for the transgene by PCR analysis of tail-tip DNA. All procedures were
conducted in accordance with protocols approved by the Animal Ethics Com-
mittee of The University of Melbourne.
Mouse inoculation with RRV. Monkey rotavirus RRV (serotype P5B, G3)
was cultivated in MA104 cells, purified by glycerol gradient ultracentrifugation,
and titrated for infectivity as described previously (23, 25). Oral inoculation was
used to mimic the natural route of infection, as is often done in studies of
rotavirus infection in rodents (12, 18, 37). Inoculation procedures were similar to
those used previously in our laboratory for NOD mice (21). In brief, after
administration of NaHCO3solution to reduce stomach acidity, mice were inoc-
ulated by gavage with 0.2 ml of 50 mM Tris-HCl buffer (pH 7.4), containing 150
mM NaCl and 5 mM CaCl2(TSC) as a virus diluent control, 1.8 ? 106fluores-
cent cell-forming units (FCFU) of RRV in TSC, or an extract of mock-infected
MA104 cells that had been harvested by freeze-thawing and clarified by low-
speed centrifugation as a control for possible cell component contamination of
purified RRV. This cell extract was diluted to the same degree as the RRV
inoculum. In some experiments (as indicated), mice were given 107FCFU of
RRV in TSC. Female and male NOD mice were inoculated at 12 and 15 weeks
of age, respectively (unless otherwise indicated) to be confident of their advanced
insulitis but avoid infection when diabetes could occur spontaneously. Similarly,
NOD8.3 TCR mice were inoculated aged 5 weeks, 1 week before naive mice first
begin to develop diabetes. Diarrhea was defined as described previously (21), and
its presence was evaluated daily for 8 days after inoculation.
Mouse inoculation with mouse rotavirus. Five-day-old BALB/c mice housed
with their rotavirus-seronegative dams in an isolation room in the animal facility
were inoculated by oral gavage as described previously (21) with 30 ?l of TSC
(controls; n ? 42) or clarified stool homogenate (10% [wt/vol] in TSC) from a
diarrheic mouse (n ? 42). In an enzyme immunoassay (EIA) using rabbit anti-
serum to RRV as capture antibody and monoclonal antibody RVA as a detector
antibody (21), this stool extract showed a mean specific optical density at 450 nm
(OD450) ? the standard deviation of 1.196 ? 0.066, indicating that a high level
of rotavirus antigen was present. According to the previously proposed nomen-
clature scheme for mouse rotaviruses, this virus is designated as the agent of
epizootic diarrhea of infant mice (EDIM)-Melbourne, abbreviated as EM here
(8). Inoculated mice were monitored for diarrhea as described above.
Detection of rotavirus in organs and samples from rotavirus-infected mice.
For analysis of RRV-infected NOD and NOD8.3 TCR mice, the small intestine,
pancreas, liver, spleen, serum, and blood cells were obtained from each animal
at days 3 to 8 after infection (n ? 4 per day). Stools were collected (one pellet
per mouse) at days 2 (n ? 20), 3 (n ? 20), 4 (n ? 20), 5 (n ? 16), 6 (n ? 12),
7 (n ? 8), and 8 (n ? 4) after infection. The procedures for collection and
processing of these tissues and stools have been described previously (21). In-
fectious RRV was detected by culture amplification of virus, followed by assay of
rotavirus antigen in cultures by capture EIA, and stools and pancreases were
examined for rotavirus antigen by EIA prior to culture, as described previously
The pancreas, liver, spleen, serum, and blood cells were collected from each
BALB/c mouse infected with mouse rotavirus EM and control BALB/c mice at
days 2 to 7 after infection (n ? 5 per day) and processed as described previously
(21). Intestinal cells were obtained by using established methods (14, 59). In
brief, the small intestine was placed in Hanks balanced salt solution containing
5% (vol/vol) fetal bovine serum (CSL, Ltd., Melbourne, Australia) and 1 mM
dithiothreitol (Sigma). Intestines were cut open lengthwise and then into 1-cm
pieces. Cells (including enterocytes) were detached by using an orbital shaker at
37°C for 40 min, filtered through a 70-?m-pore-size cell strainer, and pelleted by
centrifugation at 450 ? g for 5 min. Since EM was not culture adapted, rotavirus
antigen in organs and samples (except the small intestine) from infected BALB/c
mice was detected by capture EIA without culture amplification. The proportion
of detached intestinal cells expressing rotavirus antigen was determined by flow
cytometric analysis of methanol-fixed cells, as described previously (22).
Assay for rotavirus antibodies. Titers of rotavirus antibodies were determined
in the sera from all mice by EIA using RRV antigen, as previously described (21).
All mice were negative for serum antibodies to RRV prior to experimentation.
Seroconversion was defined as a fourfold increase in antibody titer.
Glucose homeostasis and diabetes monitoring. Glycosuria was monitored by
using Diastix reagent strips at 1 day prior to RRV or control inoculation and at
days 3, 5, 7, 9, and 11 after infection. In the weekly screening after inoculation,
urine testing was alternated with measurement of blood levels by using an
Accu-Check Advantage II blood glucose meter and strips. Blood glucose levels
were determined immediately in glycosuric mice. As described previously, con-
secutive blood glucose levels of ?240 mg/dl on two occasions 2 to 3 days apart
were considered to indicate diabetes development (21).
Histology. Pancreases were fixed in Bouin’s solution and embedded in paraffin.
Sections (5 ?m) were cut 200 ?m apart at four levels and stained with hema-
toxylin and eosin as previously described (21). For each mouse a quantitative
insulitis score was determined by using an adaptation of a previously described
method (43). All islets present in each level of the pancreas (at least 20 per
mouse) were scored as 0 (no islet infiltrate), 1 (insulitis visible around the outer
edge of the islet), 2 (intra-islet infiltration into ?30% of total islet area), 3
(intra-islet infiltration into 30 to ?70% of total islet area), or 4 (infiltration into
70 to 100% of the islet).
Isolation and flow cytometric analysis of cells from islets and PLN. Purified
islets were obtained from pancreases by bile duct cannulation, collagenase di-
gestion, and density gradient separation, as described previously (32). Cells
released from islets with trypsin-EDTA were allowed to recover in culture
medium for 0.5 to 1.5 h before analysis, as described previously (13). PLN were
dissected from harvested pancreases, mechanically disrupted by using glass
slides, filtered through nylon mesh, and washed. Single-cell suspensions from
islets and PLN were labeled for T- and B-cell analysis with combinations of
monoclonal antibodies specific to mouse antigens (BD Biosciences). Cells were
labeled with fluorescein isothiocyanate-conjugated CD4 (GK1.5), phycoerythrin-
conjugated CD45R/B220 (RA3-6B2), and CyC-conjugated CD8a (53-6.7). In
addition, islet cells were labeled with allophycocyanin-conjugated anti-CD45
(30-F11) to isolate lymphocytes from other cell types. For detection of major
histocompatibility complex class I (MHC-I), islet cells were labeled with biotin-
ylated anti-H-2Kdand allophycocyanin-conjugated anti-CD45, followed by phy-
coerythrin-conjugated streptavidin. Propidium iodide at 50 ?g/ml was included at
the final step to allow the exclusion of dead cells. Cell data were acquired on a
FACScalibur flow cytometer and analyzed with CellQuest Pro software v5.2 (BD
Biosciences). Forward and side scatter gates for lymphocytes were set on CD45?
cells from islets and on all PLN cells. These lymphocytes were then analyzed for
CD4, CD8, and B220.
Islet expression of TNF-? mRNA. Total RNA was extracted from purified
islets by using TRIzol reagent (Invitrogen). Total RNA was reverse transcribed
by using random primers. Real-time PCR was conducted using Assays-on-De-
mand kits (Applied Biosystems) for tumor necrosis factor alpha (TNF-?) and ?
actin as a reference gene, in a Corbett Research Rotor-Gene 3000 sequence
6140GRAHAM ET AL. J. VIROL.
Statistical analysis. The Student paired t test, Mann-Whitney test, and one-
way analysis of variance (ANOVA) were used as appropriate. Other tests were
used as indicated. Diabetes curves were evaluated by Kaplan-Meier life-table
analysis and the log-rank test for trend.
Older NOD and NOD8.3 TCR mice infected with RRV
showed asymptomatic infection, limited intestinal replication,
and no viremia or extraintestinal spread. In order to examine
virological parameters of rotavirus infection in insulitic mice,
NOD mice aged 12 weeks (females) or 15 weeks (males) and
NOD8.3 TCR mice aged 5 weeks were infected by oral gavage
with RRV rotavirus. All RRV-inoculated mice seroconverted
to homologous rotavirus by 2 weeks after infection, whereas all
diluent-inoculated mice showed negative antibody titers to
RRV of ?1:50 (see Fig. 3; also data not shown). No RRV-
infected or control mice showed diarrhea. On day 4 after
infection, 5% (1/20) of stools from both male and female NOD
mice contained infectious RRV, and a stool from another
female mouse contained rotavirus antigen but not infectious
virus. On day 7 after infection, 12% (1/8) of stools from fe-
males also contained RRV antigen. Males did not excrete any
detectable rotavirus antigen at any time after infection. Over-
all, 23% of female NOD mice and 5% of males excreted
detectable infectious RRV and/or rotavirus antigen. On days 2
to 4 after infection of approximately equal numbers of female
and male NOD8.3 TCR mice, stools from 23% of females and
18% of males contained infectious RRV, and 25% (1/4) of the
small intestines collected on day 4 contained infectious RRV.
Overall, RRV was found intestinally in 45% of NOD8.3 TCR
mice. The altered T-cell repertoire of NOD8.3 TCR mice
might have contributed to their increased rate of intestinal
RRV detection over NOD mice, through a reduced ability to
control RRV replication. The stool and intestinal extracts gen-
erally contained low levels of infectious RRV, showing OD460
values for RRV antigen by EIA after culture amplification of
?0.25, with one exception (OD460? 1.1). In our experience,
samples with OD460values of ?0.25 contain levels of infectious
RRV below the detection limit for direct titration in permissive
cells (4 ? 102FCFU/ml), so titration was not attempted (21).
Infectious RRV was not detected in the pancreases, livers,
spleens, sera, or blood cells of these NOD and NOD8.3 TCR
mice by culture amplification and then EIA. This method was
successfully used in our laboratory by the same experimenters
in the same year to detect infectious rotavirus at these sites in
RRV-infected infant NOD mice (21). In addition, RRV anti-
gen was not detected in the pancreas by EIA.
RRV infection of female and male NOD mice with estab-
lished pancreatic insulitis accelerated diabetes development.
To determine whether rotavirus infection modulates the tim-
ing and incidence of spontaneous diabetes onset, groups of 12-
to 15-week-old NOD mice were orally inoculated with RRV,
virus diluent, or cell extract and then monitored for glycosuria
and hyperglycemia (Fig. 1). These studies overlapped in time
and location with closely related experiments undertaken by
our group, which showed that oral RRV inoculation of NOD
mice delays diabetes onset in infants and has little effect in
adults aged 4 to 6 weeks (21). Mice inoculated with cell extract
were included to control for the effect of any contamination of
purified RRV with immunostimulatory components from cells
used to propagate RRV.
NOD mice did not show glucosuria in the 2 weeks after
RRV infection or control inoculations. However, hyperglyce-
mia and diabetes began to develop at 3 weeks (females) and 9
weeks (males) after RRV infection, 4 weeks (females) and 7
weeks (males) earlier than mice inoculated with virus diluent
Two female NOD mice inoculated with cell extract devel-
oped diabetes 1 week and 4 weeks earlier than any virus di-
luent-inoculated females (Fig. 1A). However, there was no
significant difference between the diabetes curves of females
inoculated with virus diluent or cell extract (P ? 0.73), indi-
cating that any MA104 cell components in the purified RRV
preparation would not materially affect diabetes development.
A significant acceleration in diabetes development was ob-
served from the diabetes curves of RRV-infected females com-
pared to females fed virus diluent (P ? 0.023) or cell extract
(P ? 0.019). The proportion of females with diabetes at 21
weeks of age increased from 14% (2/14) in those fed cell
extract to 60% (9/15) after RRV infection (P ? 0.021). How-
ever, by 34 weeks of age the diabetes proportions in control
and RRV-infected mice were similar (0.24 ? P ? 0.43).
The diabetes curves of cell extract- and RRV-inoculated
males showed no significant difference from those of diluent-
fed males (Fig. 1B; P ? 0.30 and P ? 0.18, respectively).
However, by survival analysis RRV-infected male mice showed
a significant acceleration in diabetes development compared to
mice fed cell extract (P ? 0.035). Compared to diluent- and
FIG. 1. Diabetes development was accelerated by RRV infection
of female (A) and male (B) NOD mice with established insulitis. Mice
were inoculated orally at 12 weeks (female) or 15 weeks (male) of age
with RRV, virus diluent, or cell extract and then monitored for dia-
betes until 34 weeks (female) or 45 weeks (male) of age. The data
provided are from a single experiment that is representative of two
independent experiments, each performed with similar numbers of
VOL. 82, 2008ROTAVIRUS ACCELERATION OF TYPE 1 DIABETES 6141
extract-fed mice, the proportion of diabetic males at 45 weeks
of age increased ?4-fold after RRV infection, from 7% (1/14)
and 0% (0/15), respectively, to 27% (4/15), although this was
not statistically significant (P ? 0.33 and P ? 0.09, respec-
RRV infection of female and male NOD8.3 TCR mice with
established pancreatic insulitis accelerated diabetes develop-
ment. The effect of RRV infection on the timing and incidence
of diabetes was examined in NOD 8.3 TCR mice, which spon-
taneously develop diabetes more rapidly than NOD mice. As
shown in Fig. 2A, the diabetes survival curves in diluent-inoc-
ulated and naive female NOD8.3 TCR mice were indistin-
guishable (P ? 0.45). Compared to diluent-inoculated and
naive females, RRV-infected females showed a highly signifi-
cant acceleration in timing of diabetes onset and increased
diabetes incidence (P ? 0.0052 and P ? 0.0072, respectively).
The increased diabetes incidence was evident 1 week after
infection and maintained to 17 weeks of age (Table 1). These
findings indicate that the kinetics of diabetes onset was accel-
erated by RRV infection.
Male NOD8.3 TCR mice showed a lower diabetes incidence
than females, as expected from previous studies and the NOD
genetic background of these mice (54). The diabetes survival
curves in diluent-inoculated and naive male NOD8.3 TCR
mice were indistinguishable (Fig. 2B; P ? 0.85). RRV infection
significantly accelerated diabetes onset and incidence in males
compared to diluent inoculation (P ? 0.038), and a trend for
infected males to show accelerated diabetes over naive males was
evident (P ? 0.06). RRV-infected and control mice showed sim-
ilar diabetes incidences at 17 weeks of age (Table 1).
Relatively high proportions of naive female (57%; 16/28)
and male (36%; 8/22) NOD8.3 TCR mice became diabetic by
17 weeks of age, and the sexes showed similar overall patterns
of diabetes development following diluent and rotavirus inoc-
ulation (Fig. 2A and B). This allowed combination of the sexes
for overall analysis, in contrast to NOD mice (Fig. 2C). The
rates of diabetes observed irrespective of sex in diluent-inocu-
lated and naive NOD8.3 TCR mice were indistinguishable (P ?
0.16). However, diabetes onset was more rapid in RRV-in-
fected mice, particularly in the first 3 weeks after infection
(Table 1). After this time the slopes of the diabetes curves of
RRV-inoculated, diluent-inoculated, and naive mice were sim-
ilar, again showing that the diabetes acceleration induced by
RRV manifested predominantly in the first 3 weeks. Overall,
RRV infection significantly accelerated the onset of diabetes
over diluent-inoculated and naive mice (P ? 0.008 by survival
analysis). The proportions of diabetic mice at 17 weeks of age
did not differ between test and control mice (Table 1). How-
ever, a subset of these mice (13 females and 19 males) was
monitored until 19 to 20 weeks of age. The diabetes incidence
in this subset increased from 47% (7/15) in diluent-inoculated
FIG. 2. Diabetes development was accelerated by RRV infection of
female (A), male (B), or female and male (C) NOD8.3 TCR mice with
or virus diluent and monitored for diabetes until 17 weeks of age. The
data were compiled from four independent experiments performed over
a 1-year period, each of which showed similar patterns of diabetes devel-
opment. The naive NOD8.3 TCR mouse curves represent spontaneous
diabetes development in the animal facility during this time.
TABLE 1. Effects of RRV infection on the timing of diabetes onset in NOD8.3 TCR mice
Time (wk) after
Proportion (%) of diabetic mice after the indicated inoculation
RRV DiluentNoneRRV Diluent NoneRRV Diluent None
aP ? 0.0005 (Fisher exact test).
bP ? 0.0035 (Fisher exact test).
c0.25 ? P ? 0.38 (Fisher exact test).
d0.059 ? P ? 0.066 (chi-square test).
6142GRAHAM ET AL. J. VIROL.
mice to 88% (15/17) in RRV-infected mice (Fisher exact test,
P ? 0.021).
Relation between serum antibody titers to RRV and degree
of diabetes acceleration. Diabetic and nondiabetic mice inoc-
ulated with RRV showed similar geometric mean titers of
serum anti-rotavirus antibodies (NOD, 1:4,300 and 1:3,200,
respectively; NOD8.3 TCR, 1:1,200 and 1:1,500, respectively;
P ? 0.05). The reduced NOD8.3 TCR mouse titers probably
resulted from their altered T-cell repertoire. Stratification of
RRV-infected mice by antibody titer demonstrated that the
extent of diabetes acceleration was significantly greater in mice
showing higher titers of anti-rotavirus antibody in serum (Fig.
3; survival analysis and log-rank test for trend, P ? 0.023 and
P ? 0.0074 for NOD, respectively, and P ? 0.042 and P ?
0.0059 for NOD8.3 TCR, respectively). High-responding NOD
and NOD8.3 TCR mice developed diabetes significantly ear-
lier than diluent-inoculated mice (Table 2; 0.017 ? P ?
0.028). NOD mouse inoculation with a fivefold-higher dose
of RRV (1.0 ? 107FCFU) did not materially affect the
diabetes curves or seroconversion antibody titers obtained
(data not shown).
Rotavirus infection of NOD mice at 10 to 20 weeks of age in
natural experiments. Several older adult NOD mice were in-
advertently exposed to EM mouse rotavirus infection when
coquarantined with mice imported from a rotavirus-positive
facility. Since EM exposure was detected during monitoring of
antibodies to RRV by EIA in sera collected fortnightly or
monthly, its timing could be determined to within a 2- to
4-week interval. No gastroenteritis was observed in the im-
ported mice or the exposed older adult NOD mice during this
period, suggesting EM was of low virulence for older adults. The
NOD mice were monitored for diabetes until 35 weeks old.
Most (6/9) of the adult male NOD mice that were exposed
to EM seroconverted to rotavirus, aged 13 to 20 weeks, and 5
of 6 (81%) of these EM-exposed mice developed diabetes aged
a mean ? the standard deviation of 25 ? 2 weeks (Fig. 4). In
contrast, none (0/3) of the EM-exposed male mice that did not
seroconvert developed diabetes (Fisher exact test, P ? 0.04).
The male diabetes incidence in this facility is ?10% at 30
weeks (Fig. 1), highlighting the extent of the increased diabetes
incidence after rotavirus seroconversion. Females that had se-
roconverted to rotavirus after oral RRV inoculation at 4 weeks
of age also were inadvertently exposed to EM. Many of these
females (6/14; 43%) seroconverted to rotavirus a second time
at 10 to 14 weeks of age. These mice all developed diabetes, at
18 ? 2 weeks of age (Fig. 4). In contrast, only 2 of 8 (25%) of
the RRV-inoculated females that did not seroconvert a second
time developed diabetes, at 16 and 25 weeks of age (P ? 0.01).
The diabetes incidence in female NOD mice in this facility was
?60% at 30 weeks (Fig. 1). These findings of diabetes accel-
eration in highly insulitic NOD mice after EM rotavirus expo-
sure are consistent with the results obtained from the RRV
infection experiments described above that were conducted in
NOD mice under controlled conditions.
All BALB/c mouse pups inoculated with a stool extract con-
taining high levels of EM antigen showed diarrhea that re-
TABLE 2. Relation between serum antibody titers to RRV and
mouse age at diabetes onset
Mean age (wk) ? SD at diabetes onset of mice with
antibody titers to RRV of:
24.2 ? 5.1
11.5 ? 2.9
22.9 ? 4.3
9.7 ? 3.6
19.3 ? 3.2c
6.5 ? 1.0d
10.6 ? 2.3
bRRV-infected NOD mice did not show titers in this range.
cP ? 0.027 compared to diluent-fed mice.
dP ? 0.018 compared to diluent-fed mice.
FIG. 3. Diabetes acceleration was of greater extent in mice showing
higher titers of anti-rotavirus antibody (Ab) in serum. The diabetes
curves for RRV-infected female NOD mice from Fig. 1 (A) and
NOD8.3 TCR mice from Fig. 2 (B) were stratified into mice with low
(1:100 to 1:400), medium (1:800 to 1:3,200), and high (1:6,400 to
1:25,600) serum antibody titers to RRV at 2 weeks after infection.
Serum from one NOD8.3 TCR mouse was unavailable for antibody
testing, so the results from that mouse were excluded.
FIG. 4. Effect of exposure to mouse rotavirus EM in a natural
experiment on diabetes development in NOD mice. Mice are stratified
into those that seroconverted to rotavirus after possible exposure to
EM (?serocon) and those that did not seroconvert (?serocon). Fe-
males had been inoculated with RRV at 4 weeks of age prior to EM
exposure, whereas males had been inoculated at 4 weeks of age with
control cell extract but had no previous rotavirus exposure.
VOL. 82, 2008 ROTAVIRUS ACCELERATION OF TYPE 1 DIABETES6143
solved without mortality and rotavirus antigen in intestinal
cells for 6 days after inoculation. Up to 6% of intestinal cells
contained antigen (Table 3). No rotavirus antigen was detected
by direct EIA in the pancreas, liver, spleen, serum, or blood
cell preparations. These data indicate that EM replicated ef-
ficiently in the small intestine without any detectable extraint-
Effect of RRV infection on pancreatic insulitis in NOD mice.
The ability of RRV infection to accelerate diabetes develop-
ment in NOD mice suggested that islet insulitis also might be
increased after RRV infection. The degree of insulitis was
scored at a range of times after RRV infection or virus diluent
inoculation of female and male NOD mice (Fig. 5).
As expected due to the ongoing diabetic process, insulitis
scores in cell extract-inoculated female mice significantly in-
creased between 13 and 17 weeks of age (Fig. 5A; P ? 0.017).
A similar effect was seen in RRV-infected females (Fig. 5A;
P ? 0.039), indicating that RRV infection did not inhibit on-
going insulitis development. No significant difference in insu-
litis scores between RRV-infected and cell extract-inoculated
females was observed at 13 or 17 weeks (P ? 0.43 and P ? 0.76,
respectively). However, the upper range of insulitis scores was
increased 1 week after RRV infection over cell extract-inocu-
lated mice at the same time (Fig. 5A), suggesting a possible
trend for RRV-infected females to show higher scores than
control mice. This is consistent with the accelerated diabetes
onset in these mice. Insulitis scores at 13 weeks of age also
were determined for females previously inoculated with RRV
or cell extract at 5 days of age (Fig. 5A). Consistent with our
previous data (21), RRV infection at 5 days of age did not
significantly alter insulitis scores (P ? 0.39). There was no
significant difference in the degree of insulitis at 13 weeks of
age between mice inoculated with the control preparation at 5
days or 12 weeks (P ? 0.61). However, females previously
infected with RRV aged 12 weeks showed significantly ele-
vated insulitis at 13 weeks of age compared to those of the
same age that were inoculated with RRV at 5 days of age (Fig.
5A; P ? 0.036).
No significant differences in scores between RRV-infected
and control male NOD mice were evident at 16, 20, or 52
weeks of age (Fig. 5B; P ? 0.51, P ? 0.49, and P ? 0.24,
respectively). However, insulitis scores in RRV-infected males
increased significantly between 1 and 5 weeks (P ? 0.048) and
1 and 37 weeks after infection (P ? 0.028). In contrast, mean
insulitis scores in control mice did not alter from 1 to 37 weeks
after infection (0.38 ? P ? 0.69). Several mice analyzed at 52
weeks showed insulitis scores similar to those of diabetic males,
but mice analyzed earlier generally showed lower scores, as
Overall, RRV infection did not inhibit insulitis development
in older female or male NOD mice, and evidence of a possible
trend for increased insulitis after RRV infection was found for
Analysis of lymphocytes in islets and PLN following RRV
infection of NOD mice. To further analyze the insulitis follow-
ing RRV infection of older NOD mice, the proportions of
CD4?T cells, CD8?T cells, and B cells infiltrating islets and
trafficking through the PLN were examined in female and male
NOD mice (Tables 4 and 5). There were no significant differ-
ences in the proportions of CD4?T cells, CD8?T cells, and B
cells in islets or PLN between RRV-infected and cell extract-
inoculated female mice (P ? 0.05). However, RRV infection of
male mice significantly increased the proportion of CD8?T
cells in islets at 1 week after infection compared to cell-extract-
inoculated mice (P ? 0.039; Table 4). B-cell proportions in
PLN and islets were significantly increased over controls at 3
weeks and 5 weeks after RRV infection of males, respectively
(P ? 0.005 and P ? 0.048, respectively; Tables 4 and 5). The
FIG. 5. Effects of RRV infection on insulitis development in NOD
mice. (A) The pancreases of female NOD mice inoculated with RRV
or virus diluent at 12 weeks of age were scored for insulitis at 1 and 5
weeks after infection, at the ages of 13 and 17 weeks, respectively.
These data are compared to the insulitis scores at a similar age (13
weeks) in the pancreases of female NOD mice inoculated by oral
gavage at 5 days of age. Nondiabetic mice only were included. (B) Pan-
creatic insulitis scores at 16, 20, and 52 weeks of age in male NOD mice
inoculated with RRV or diluent at 15 weeks of age that had not
become diabetic. As positive controls, the pancreas was collected at
diabetes onset from two male NOD mice (one RRV inoculated) and
analyzed for insulitis. All RRV-infected mice showed serum antibody
titers of ?1:50 and 1:3,200 at 1 and 5 weeks after infection, respec-
tively. The bars indicate the means.
TABLE 3. Intestinal replication of mouse rotavirus EM in infant
Time (days) after
Mice showing rotavirus
antigen in intestinal
antigen (mean % ?
3.4 ? 0.6
5.9 ? 0.3
5.6 ? 0.6
1.6 ? 0.8
0.1 ? 0.1
0.0 ? 0.00
6144 GRAHAM ET AL.J. VIROL.
CD4?/CD8?T-cell ratios also were determined for these mice
(Fig. 6). In the first 8 weeks after RRV infection, CD4?/CD8?
ratios in islets and PLN of female NOD mice were unaltered
compared to extract-inoculated mice (Fig. 6; P ? 0.05). How-
ever, CD4?/CD8?ratios in islets decreased significantly in
male NOD mice 1 week after RRV infection compared to
extract-fed mice (Fig. 6; P ? 0.003). Overall, these data indi-
cate that T-lymphocyte proportions in islets and PLN were
unaltered in female NOD mice after RRV infection. However,
RRV infection in male NOD mice led to increased CD8?
T-cell proportions and reduced CD4?/CD8?ratios in islets at
1 week after infection.
MHC-I expression on ? cells was increased after RRV in-
fection in NOD8.3 TCR mice. Upregulation of MHC-I on ?
cells has been proposed to be important for triggering progres-
sion to diabetes (17). The ability of rotavirus infection to mod-
ify MHC-I expression by ? cells was analyzed. As shown in Fig.
7, MHC-I levels increased significantly over time in both mock-
and RRV-infected NOD8.3 TCR mice (ANOVA post test for
linear trend, P ? 0.0003). RRV infection produced signifi-
cantly increased MHC-I levels on NOD8.3 TCR ? cells at day
10 after infection compared to mock-infected mice on the
same day (P ? 0.012). MHC-I levels in female NOD mice were
examined at days 1 to 5, 7, 14, and 35 after mock or RRV
infection. In female NOD mice, ? cell MHC-I expression in-
creases with age more slowly than NOD8.3 TCR MHC-I (48).
Consistent with this, ? cell MHC-I levels did not alter signifi-
cantly over the study period in mock- or RRV-infected mice
(data not shown; ANOVA post test for linear trend, P ? 0.05).
MHC-I levels in RRV-infected female NOD mice were similar
to those in mock-infected mice on the same day (data not
shown; P ? 0.05). Thus, alteration in MHC-I levels was not
detected in the 35 days following RRV infection of female
TABLE 4. Proportions of lymphocyte subsets in islets of NOD mice inoculated with cell extract or RRV
Time (wk) after inoculationa
Mean % of lymphocytes ? SEM in islets with the indicated cell surface markerb
Cell extractRRVCell extractRRV Cell extractRRV
1 (n ? 10)
2 (n ? 8)
5 (n ? 9)
8 (n ? 8)
38.5 ? 2.6
38.2 ? 1.4
44.0 ? 1.6
39.0 ? 2.8
36.6 ? 1.5
38.1 ? 1.9
44.2 ? 2.7
32.8 ? 1.0
17.9 ? 1.1
20.2 ? 0.5
18.1 ? 1.3
18.2 ? 1.3
18.6 ? 1.3
21.3 ? 1.4
20.2 ? 0.9
17.5 ? 1.9
25.0 ? 1.4
23.2 ? 1.5
23.9 ? 2.1
21.3 ? 2.4
26.8 ? 2.2
23.8 ? 1.9
20.6 ? 3.2
27.9 ? 4.4
1 (n ? 19)c
3 (n ? 7)
5 (n ? 7)
7 (n ? 6)
10 (n ? 5)
36.1 ? 2.3
39.8 ? 1.2
42.6 ? 2.1
33.6 ? 2.5
31.8 ? 2.0
32.3 ? 1.6
39.5 ? 2.1
37.7 ? 1.5
36.8 ? 2.2
35.5 ? 2.7
16.1 ? 2.1
14.2 ? 1.3
18.4 ? 0.7
16.6 ? 1.2
11.2 ? 0.8
21.4 ? 1.3d
17.4 ? 1.0
15.4 ? 1.4
18.4 ? 1.3
13.2 ? 1.0
30.1 ? 2.3
27.3 ? 0.9
23.6 ? 1.4
31.3 ? 2.3
35.0 ? 3.4
30.2 ? 2.1
28.4 ? 3.1
29.8 ? 2.2e
26.7 ? 2.1
31.6 ? 3.5
an, Number of animals tested.
bLymphocyte subsets showing significant differences in proportions are indicated in boldface.
cExcept for the cell extract group, where n ? 11.
dP ? 0.039.
eP ? 0.048.
TABLE 5. Proportions of lymphocyte subsets in PLNs of NOD mice inoculated with cell extract or RRV
Time (wk) after inoculationa
Mean % of lymphocytes ? SEM in the PLN with the indicated cell surface markerb
Cell extract RRVCell extract RRVCell extract RRV
1 (n ? 10)
2 (n ? 7)
5 (n ? 9)
8 (n ? 8)
40.3 ? 2.3
35.5 ? 1.3
33.8 ? 2.0
38.1 ? 1.4
42.2 ? 2.1
38.3 ? 2.6
37.1 ? 1.3
38.5 ? 2.4
17.1 ? 0.8
15.7 ? 0.8
14.9 ? 1.5
16.9 ? 0.9
17.1 ? 0.8
15.1 ? 0.8
13.4 ? 1.6
17.1 ? 0.7
9.7 ? 1.5
11.2 ? 1.6
12.0 ? 1.4
9.7 ? 1.5
12.1 ? 1.6
7.6 ? 1.1
10.9 ? 1.7
9.8 ? 2.0
1 (n ? 12)c
3 (n ? 7)
5 (n ? 7)
7 (n ? 6)
10 (n ? 5)
35.6 ? 2.5
27.0 ? 2.4
24.3 ? 2.1
23.4 ? 5.0
36.2 ? 2.2
32.9 ? 2.0
28.6 ? 2.7
28.4 ? 4.2
29.7 ? 3.8
39.6 ? 1.5
16.1 ? 1.0
18.0 ? 2.9
13.7 ? 1.0
14.5 ? 2.1
15.9 ? 1.3
14.6 ? 0.7
14.4 ? 1.7
15.0 ? 2.1
16.6 ? 1.7
18.6 ? 0.3
12.2 ? 2.9
7.1 ? 0.3
11.8 ? 3.9
10.1 ? 1.5
10.9 ? 1.7
12.1 ? 1.4
11.2 ? 1.0d
9.2 ? 1.7
11.7 ? 1.6
12.0 ? 0.9
an, Number of animals tested.
bLymphocyte subsets showing significant differences in proportions are indicated in boldface.
cExcept for the cell extract group, where n ? 5.
dP ? 0.0047 (Student t test with Welch’s correction).
VOL. 82, 2008ROTAVIRUS ACCELERATION OF TYPE 1 DIABETES6145
Islet TNF-? levels in insulitic mice were increased by RRV
infection. Proinflammatory cytokines such as TNF-? are cyto-
toxic to islets. TNF-? mRNA levels were determined in iso-
lated islets of mock- and RRV-infected mice (Fig. 8). RRV
infection significantly increased TNF-? mRNA levels at 10 to
15 days postinfection in NOD8.3 TCR mice (P ? 0.036), and
7 to 14 days in female NOD mice (P ? 0.0033). TNF-? mRNA
levels were unchanged by RRV infection at other times.
Experiments presented here show that RRV rotavirus infec-
tion of diabetes-prone mice with established insulitis acceler-
ates diabetes development. Thus, rotavirus can hasten diabetes
onset once ?-cell autoimmunity is established. This is the first
described study in an animal model suggesting a relationship
between rotavirus infection and diabetes exacerbation. These
findings support the proposed association of rotavirus infection
with increased islet autoimmunity in children (26). Taken in
conjunction with our previous demonstration that RRV infec-
tion in preinsulitic NOD mice can delay diabetes development
(21), these new results indicate that the timing of RRV infec-
tion in relation to mouse age and degree of insulitis determines
whether diabetes onset is accelerated, delayed, or unaltered.
The specificity of the diabetes acceleration for RRV is high-
lighted by the lack of diabetes modulation by control cell ex-
tracts, and the overlap in time and place of execution between
the accelerated diabetes curves after RRV infection in older
adult mice described here and those showing diabetes delay in
FIG. 6. Effects of RRV infection on ratios of CD4?to CD8?T cells in islets and PLN of NOD mice. At each time point, at least 5,000
lymphocyte-sized cells were analyzed for each cell marker (as described in Materials and Methods) from the groups of mice described in Tables
4 and 5 that had not become diabetic. All RRV-inoculated mice seroconverted by 2 weeks after infection. Mice showed a geometric mean serum
antibody titer of 1:2,800 at 5 weeks after infection. Error bars represent the standard error of the mean. A significant difference in ratios between
RRV-infected and control mice is marked with a star (P ? 0.003).
FIG. 7. Expression of MHC-I on ? cells of NOD8.3 TCR mice
after RRV infection. Viable, CD45-negative islet cells were identified
as ? cells by their autofluorescence, as previously described (13).
Groups of five to seven nondiabetic mice were analyzed at each time
point. Mice showed geometric mean serum antibody titers to RRV of
1:110 and 1:730 at days 10 and 15 after RRV inoculation, respectively.
FIG. 8. Levels of islet TNF-? mRNA following RRV infection of
insulitic mice. Groups of four to six mock- and RRV-inoculated non-
diabetic mice were analyzed at each time point. The data from con-
secutive time points were merged for simplicity. The data for RRV-
infected mice are presented relative to the mean mRNA level for
mock-infected mice at the same time, which was standardized to 1.0
(dotted line). Significant differences between RRV- and mock-infected
mice are marked with stars (0.0033 ? P ? 0.036). Mouse serum
antibody titers to RRV were similar to those described in the legends
to Fig. 6 and 7.
6146 GRAHAM ET AL.J. VIROL.
younger adult NOD mice infected with RRV under similar
conditions (21). Apart from the test variable of mouse age
(corresponding to the extent of insulitis), the only material
difference between our earlier studies and those reported here
is the RRV dose. Younger adult mice received a 10-fold-higher
dose (2 ? 107FCFU) than older adult mice (1.8 ? 106FCFU)
(21). However, we determined that inoculation of female NOD
mice at 12 weeks of age with 107FCFU of RRV did not
materially affect the diabetes curves or seroconversion anti-
body titers obtained (data not shown). Preexisting severe in-
sulitis or possibly other age- and insulitis-related events are
important for the diabetes exacerbation caused by RRV.
RRV infection accelerates diabetes onset in all NOD and
NOD8.3 TCR mice. The greater effect in females is consistent
with their higher incidence of spontaneous diabetes (3, 54).
Our diabetes survival curves indicate that RRV has a more
pronounced effect on diabetes development in NOD8.3 TCR
mice than NOD mice. Also, infection increases diabetes inci-
dence in female NOD8.3 TCR mice, in female and male
NOD8.3 TCR mice combined, and transiently in female NOD
mice. Thus, RRV affects the timing of diabetes onset to a
greater extent than incidence. The effect of RRV on diabetes
manifests mainly in first 3 to 9 weeks after infection, suggesting
that immune responses to infection are directly involved in
diabetes modulation by RRV.
Extrapolation of findings from NOD mice to humans can be
problematic. However, this model is highly relevant to human
disease (3, 40, 46). The timing of rotavirus infection in relation
to the prediabetic stage of at-risk children might similarly affect
the outcome for their autoimmunity. This could help resolve
the apparently conflicting findings regarding rotavirus in-
fection and diabetes in Australian and Finnish children (6, 26).
The majority (22 of 24; 92%) of the Australians were moni-
tored to an older age (2.5 to 7 years) than the Finns (2 years)
and so would have been more likely to encounter rotavirus at
an advanced prediabetic stage during the study. In a further
parallel, most children in the advanced prediabetic stage ex-
hibited mild gastroenteritis or asymptomatic infection upon
rotavirus reexposure. Similarly, mice with diabetes accelera-
tion were asymptomatically infected with RRV.
T-cell molecular mimicry of rotavirus VP7 with HLA recog-
nition regions in human GAD and IA-2 was proposed to ex-
plain the possible association between islet autoimmunity and
rotavirus infection in children (26, 27). However, no corre-
sponding mimicry has been demonstrated between murine de-
terminants of MHC binding and rotavirus. Mimicry between
rotavirus and these autoantigens in NOD mice is unlikely to
occur since the critical human genes HLA-DR and HLA-DQ
that confer diabetes susceptibility are absent (53). Any role for
mimicry in these mice or humans was therefore not assessed in
our experiments. However, molecular mimicry alone is consid-
ered to be unlikely to be sufficient to cause diabetes (10, 19).
Consistent with this, our demonstration that RRV accelerates
diabetes onset in NOD mice suggests that rotavirus mimicry
with GAD and IA-2 might not be critical for rotavirus modu-
lation of diabetes progression. Islet infiltration of diabetogenic
CD8?T cells expressing the 8.3 TCR occurs early in the
disease process in NOD8.3 TCR mice (54). Many CD8?T
cells recruited to NOD mouse islets also recognize IGRP (2,
52). When activated with the cognate mimotope derived from
IGRP (NRP), these cells respond to numerous NRP peptide
analogues. No NRP mimics were identified in rotavirus pro-
teins through protein database searches, so it is most unlikely
that NRP molecular mimicry is involved in the diabetes exac-
erbation by RRV (1; B. S. Coulson, unpublished data).
The degree of diabetes acceleration and extent of intestinal
RRV detection both followed the pattern NOD8.3 TCR ?
female NOD ? male NOD. This implies a relation might exist
between these parameters. Studies with a range of 50% virus
shedding doses would help address this issue. Rotavirus infec-
tion in mice with invasive insulitis did not involve the pancreas,
so direct pancreatic infection is not involved in RRV-induced
diabetes acceleration. Possible mechanisms for ?-cell damage
induced by other potentially diabetogenic viruses (e.g., group B
coxsackieviruses [CVB]), including cytolytic infection and de-
struction by antiviral immune responses, are thus unlikely to be
relevant to RRV (19, 28, 50, 53). In contrast to RRV, pancre-
atic CVB infection is necessary for diabetes acceleration in
insulitic female NOD mice (16, 43). In spite of this key differ-
ence in tropism, RRV and CVB show several parallel effects.
Both viruses replicate in cultured islets but show a more re-
stricted pancreatic involvement in vivo in virus-inoculated
mice, and their role in type 1 diabetes relates to other factors
apart from virus infection (11, 50; the present study). Notably,
RRV and CVB show similar age-dependent effects on diabetes
curves in female NOD mice (16, 43, 44, 51). Inoculation aged
4 weeks with diverse CVB strains protects against diabetes,
whereas infection aged 12 weeks with virulent CVB at a low
dose, or CVB of low virulence at a high dose, accelerates
diabetes (50). It will be important to determine the effect of
rotavirus dose on diabetogenicity.
Since RRV is not pancreatotropic in these older mice, al-
ternative immunological mechanisms of diabetes exacerbation,
such as those involving virus-induced proinflammatory media-
tor secretion, might be operating. These would be consistent
with the observed relation between the level of serum antibody
response to RRV and the degree of diabetes acceleration in
insulitic mice. In addition to the degree of intestinal replication
(see above), the strength of the murine immune response to
rotavirus is a likely predictor of accelerated diabetes onset.
This relation between rotavirus antibody titer and diabetes
acceleration in NOD8.3 mice also suggests that CD8?T cells
are involved. This is supported by the increased proportions of
CD8?T cells in islets of male NOD mice after RRV infection.
It is proposed that increased CD8?T-cell activity might pro-
duce the increased levels of islet TNF-? mRNA observed here
after RRV infection. TNF-? induces MHC-I expression on ?
cells (9). The simultaneous increases in TNF-? mRNA and
MHC-I at 10 days after RRV infection of NOD8.3 TCR mice
imply that these could be functionally linked. It has been pro-
posed that increased ?-cell MHC-I creates a “fertile field” for
the immune system by making ? cells more recognizable (19).
In the lymphocytic choriomeningitis virus model of virus-in-
duced diabetes, MHC-I upregulation plays a central role in ?-
cell death (42). Thus, ?-cell MHC-I upregulation might be
important for RRV-induced diabetes acceleration. This could
lead to increased immune surveillance, destruction of ? cells
and accelerated diabetes (9, 17, 19). TNF-? is elevated in
VOL. 82, 2008ROTAVIRUS ACCELERATION OF TYPE 1 DIABETES6147
acute-phase serum from children with rotavirus gastroenteritis
(30), and rapidly induced in the serum and intestine following
human rotavirus infection in the pig model (5).
As for infant and young adult NOD mice, RRV infection in
older NOD mice did not inhibit ongoing insulitis development
(21). The trend for increased insulitis in older female and male
NOD mice after RRV infection (Fig. 5) supports the acceler-
ated diabetes observed in these mice and contrasts with the
unaltered or slightly reduced insulitis in female NOD mice
infected orally as infants or young adults, respectively (21).
Overall, under conditions producing diabetes acceleration,
RRV infection results in a trend for increased insulitis in NOD
mice. Conversely, when RRV infection delays diabetes in
NOD mice, little or no insulitis reduction is observed. The
trend for increased insulitis in older female NOD mice after
RRV infection was unrelated to the proportions of the CD4?
and CD8?T lymphocytes present in the insulitis lesion. It will
be important to determine the activation status of the major
infiltrating cell types within the lesion. In male NOD mice, the
trend for increased insulitis and islet CD8?T-cell proportions
after RRV infection suggest that these cells might play a role
in the diabetes acceleration observed. These sex-related differ-
ences in the effect of RRV on the composition of the insulitic
lesion probably relate to the higher insulitis scores in RRV-
infected NOD females than males at 16 to 17 weeks of age
(Fig. 5). Any effect of RRV infection on components of the
insulitic lesion might depend on the particular degree and
nature of insulitis present at the time of infection.
Based on antigen detection, replication of EM mouse rota-
virus was restricted to the intestine in infant BALB/c mice,
suggesting that EM is less able to spread extraintestinally than
other EDIM strains, including the original strain EW (7, 8). On
days 3 to 5 after infection between 2 and 6% of the intestinal
cells contained rotavirus antigen in our studies. These propor-
tions are similar to those found previously (?1 to ?19%) in
EW-infected infant BALB/c mice (39, 45). This suggests that
EM replicates intestinally to an extent similar to that of EW.
Although mouse numbers were small, EM exposure of older
NOD mice was associated with diabetes of accelerated onset
and increased incidence, an observation consistent with our
findings in RRV-infected older mice. In the lymphocytic cho-
riomeningitis virus mouse model, sequential infection with re-
lated arenaviruses is necessary to convert insulitis to overt
diabetes (10). Older adult mice were infected once with RRV
in our controlled experiments. However, serendipitous sequen-
tial infection of female NOD mice with RRV at 4 weeks of age
and then EM as older adults also was associated with acceler-
ated diabetes onset. This is notable since RRV infection at 4
weeks of age alone has no effect on diabetes incidence (21).
Further studies of mouse rotavirus effects and the importance
of sequential rotavirus infections are needed.
Our findings indicate that RRV rotavirus exacerbates mu-
rine diabetes by a mechanism that does not depend on direct
pancreatic infection. Worldwide implementation of childhood
vaccination with live attenuated rotaviruses is currently under
way (4). The new animal models described here will be key
tools in determining the effects of human rotaviruses and ro-
tavirus vaccine strains on diabetes development.
We are grateful to Pere Santamaria for provision of the NOD8.3
TCR mouse and to Helen Thomas and Nadine Dudek for advice on
islet isolation and analysis. Fiona E. Fleming, Gavan Holloway, Peter
Halasz, Nicole Webster, Jessica Pane, and Stacey Fynch provided
excellent technical assistance. We thank David Taylor and Rhiannon
Hall for mouse husbandry.
This study was supported by project grants (208900 and 509008) and
research fellowship grants (172305, 251546, 299861, and 350253) from
the National Health and Medical Research Council of Australia and
the Melbourne Research Grants Scheme of The University of Mel-
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