Development of a reverse-genetics system for
murine norovirus 3: long-term persistence occurs in
the caecum and colon
Armando Arias, Dalan Bailey,3 Yasmin Chaudhry and Ian Goodfellow
Received 19 February 2012
Accepted 11 April 2012
Section of Virology, Department of Medicine, Imperial College London, Norfolk Place, London,
W2 1PG, UK
Human noroviruses (HuNoV) are a major cause of viral gastroenteritis worldwide, yet, due to the
inability to propagate HuNoV in cell culture, murine norovirus (MNV) is typically used as a
surrogate to study norovirus biology. MNV-3 represents an attractive strain to study norovirus
infections in vivo because it establishes persistence in wild-type mice, yet causes symptoms
resembling gastroenteritis in immune-compromised STAT1”/”mice. The lack of reverse-genetics
approaches to recover genetically defined MNV-3 has limited further studies on the identification
of viral sequences that contribute to persistence. Here we report the establishment of a combined
DNA-based reverse-genetics and mouse-model system to study persistent MNV-3 infections in
wild-type (C57BL/6) mice. Viral RNA and infectious virus were detected in faeces for at least
56 days after inoculation. Strikingly, the highest concentrations of viral RNA during persistence
were detected in the caecum and colon, suggesting that viral persistence is maintained in these
tissues. Possible adaptive changes arising during persistence in vivo appeared to accumulate in
the minor capsid protein (VP2) and the viral polymerase (NS7), in contrast with adaptive mutations
selected during cell-culture passages in RAW264.7 cells that appeared in the major capsid
protein (VP1) and non-structural protein NS4. This system provides an attractive model that can
be readily used to identify viral sequences that contribute to persistence in an immunocompetent
host and to more acute infection in an immunocompromised host, providing new insights into the
biology of norovirus infections.
Gastroenteritis remains one of the top five causes of death
worldwide and the second most common cause in low-
income countries (Monroe, 2011). The development of a
rotavirus vaccine places human noroviruses (HuNoV) as
the leading cause of food-borne disease and non-bacterial
gastroenteritis worldwide (Kahan et al., 2011; Koo et al.,
2010). It has been estimated that HuNoV infections in de-
veloping countries result in .200000 deaths and .900000
hospitalizations of children younger than 5 years every year
(Patel et al., 2008). In high-income countries, however, low
mortality rates are reported for HuNoV, with the major
impact being observed on elderly and immunocompro-
mised patients. It is estimated that 23 million symptomatic
infections occur each year in the USA alone, resulting in at
least 300 deaths (Harris et al., 2008; Mead et al., 1999; van
Asten et al., 2011). Frequent large outbreaks caused by
HuNoV are reported during the winter season, affecting
the operational capacity of many closed environments (e.g.
schools, hospitals, military camps). Large economic losses
(estimated to be .£100 million in the UK alone) are
therefore associated with hospital disruptions caused by
HuNoV outbreaks (Lopman et al., 2004). In addition to
acute gastroenteritis, noroviruses have recently been
linked to a number of significant clinical diseases, such
as the exacerbation of inflammatory bowel disease (IBD)
(e.g. Crohn’s disease, ulcerative colitis), seizures in infants
and other neurological disorders (Byrnes & Griffin, 2000;
CDC, 2002; Chen et al., 2009; Ito et al., 2006; Khan et al.,
2009). Recent studies carried out with animal models have
supported the connection between norovirus infection
and the exacerbation of IBD (Cadwell et al., 2010; Lencioni
et al., 2008).
The lack of efficient systems to cultivate HuNoV in cell
culture has limited studies on norovirus replication and
pathogenesis. Discovery of the closely related murine
norovirus (MNV), capable of replication in several cell
lines, has provided an ideal alternative with which to study
the molecular biology of noroviruses and the identification
of antiviral strategies to control them (Karst et al., 2003;
3Present address: Institute for Animal Health – Pirbright, Ash Rd,
Guildford, GU24 0NF, UK.
The GenBank/EMBL/DDBJ accession number for the MNV-3 cDNA
sequence used here to recover infectious MNV-3 by reverse genetics is
Journal of General Virology (2012), 93, 1432–1441
1432042176G2012 SGMPrinted in Great Britain
Wobus et al., 2004). MNV-1 was the first strain to be
isolated and was found to lead to a rapid systemic and
lethal infection in immunocompromised STAT12/2mice
(Karst et al., 2003). MNV-1 replicates in both immuno-
compromised and wild-type mice, although in the presence
of a competent immune system, virus replication is rapidly
controlled and cleared, becoming undetectable in faeces
and organs from 7 days post-infection (Hsu et al., 2006;
Kahan et al., 2011; Karst et al., 2003; Mumphrey et al.,
2007). Following the identification of MNV-1, three new
MNV strains were isolated from asymptomatic mice from
different geographical location colonies, referred to as
MNV-2, -3 and -4. These three new strains were able to
establish persistent infections in wild-type ICR (imprint-
ing control region) mice that lasted for .10 weeks and
resulted in large amounts of viral RNA being shed in the
faeces (Hsu et al., 2006). Subsequent studies confirmed that
MNV is highly prevalent in research colonies and that the
different strains isolated are genetically closely related and
form a single serotype (Barron et al., 2011; Hsu et al., 2007;
Thackray et al., 2007).
The MNV genome is a positive-stranded RNA molecule
around 7.5 kb in length, containing four ORFs (Fig. 1a).
Viral RNA replication, catalysed by the viral RNA-
dependent RNA polymerase NS7, results in the generation
of new viral genomes, as well as a subgenomic RNA
encompassing the ORF2–ORF4 coding region. Replication
is primed by VPg (NS5), which also plays a key role in
recruiting host factors to initiate VPg-dependent viral
protein synthesis (Chaudhry et al., 2006; Daughenbaugh
et al., 2003, 2006; Goodfellow et al., 2005). Translation of
ORF-1 leads to the synthesis of a large polyprotein that is
cleaved into the different non-structural mature proteins
(NS1–7) (Sosnovtsev et al., 2006). Translation of the
subgenomic RNA leads to production of the capsid pro-
teins VP1 and VP2 (ORF2 and 3, respectively), as well as
the innate immune antagonist VF1 (ORF4) (McFadden
et al., 2011; Wobus et al., 2006).
The development of several reverse-genetics approaches for
MNV has allowed molecular characterization of the viral and
host factors that regulate norovirus replication (Chaudhry
et al., 2007; Ward et al., 2007; Yunus et al., 2010). We have
shown previously that genetically defined wild-type MNV-1
and mutant viruses recovered in cell culture can establish an
acute infection in mice, which has been instrumental for the
molecular characterization of MNV-1 in cell culture and
in vivo (Bailey et al., 2008, 2010; McFadden et al., 2011). The
recent identification of other MNV strains able to establish
stable persistent infections in mice has opened new avenues
to understanding viral sequences that may contribute to
viral persistence. However, the absence of reverse-genetics
approaches for these new MNV variants has limited the
24 h48 h
pT7 : MNV-1
pT7 : MNV-3
pT7 : F/S
Fig. 1. Recovery of MNV-3 by reverse genetics. (a) Schematic representation of the MNV-3 genomic and subgenomic RNAs.
MNV-3 contains four ORFs. Translation of ORF1 results in the synthesis of a viral polyprotein that is then processed
proteolytically into different mature non-structural viral proteins as indicated. Translation of the subgenomic RNA results in the
synthesis of major capsid protein VP1 (ORF2), minor capsid protein VP2 (ORF3) and virulence factor VF1 (ORF4). The
sequence of pT7:MNV-3 differs from the previously reported sequence in 53 nt positions along the genome, represented as
short arrows with non-filled arrowheads (synonymous) and as long arrows with filled arrowheads (non-synonymous). (b) TCID50
virus titres obtained after recovery of pT7:MNV-1, pT7:MNV-3 and pT7:F/S (pT7:MNV with a frameshift mutation) in BSR-T7
cells 24 and 48 h post-transfection, as explained in the text. (c) Virus titres of MNV-3 samples resulting from serial passage of
MNV-3 recovered in RAW264.7 cells. MNV-3 recovered in (b) was used to infect RAW264.7 cells at a low m.o.i. (0.001).
MNV-3 produced (passage 1) was used for serial passage at a low m.o.i. of 0.1 (passages 2–5) or 0.3 (passage 6) in
Persistent animal model for murine norovirus 3
establishment of an animal model to study persistent
infections in mice with a genetically defined MNV isolate.
Here, we describe for the first time the combination of
reverse-genetics approaches and animal models to study
the establishment of a persistent norovirus infection in
wild-type C57BL/6 mice. Viral RNA was detected in faeces
for at least 56 days. In addition, all infected animals dis-
played high MNV-3 RNA loads in the caecum and colon,
suggesting that these are the major sites for persistent
Reverse-genetics recovery of MNV-3
To enable the generation of a full-length cDNA clone of
MNV-3, a sample of MNV-3 provided by Robert Livingston
(Hsu et al., 2007) was used to infect RAW264.7 cells and
total RNA was extracted 24 h post-infection as explained in
Methods. This collected sample represented the viral
population after three rounds of infection at low m.o.i. in
RAW264.7cells. Thefull viral genome was then amplified by
RT-PCR using primers that introduced a truncated T7 RNA
polymerase promoter at the 59 end and a tail of 26 adenines
at the 39 end. Cloning of the amplified fragment into the
backbone plasmid used previously to generate an MNV-1
reverse-genetics system (Chaudhry et al., 2007) resulted
in the insertion of a self-cleaving d-ribozyme sequence after
the A26sequence to ensure the generation of a defined 39
end (Walker et al., 2003). A number of constructs were
sequenced in their entirety and screened for their ability to
produce infectious virus (data not shown). Surprisingly, the
consensus sequence and the sequences of three individual
constructs displayed .50 common sequence alterations
with respect to the initially reported MNV-3 sequence (Hsu
et al., 2007).
We selected pT7:MNV-3 clone 2 as our reference clone,
referred to here as pT7:MNV-3 (GenBank accession no.
JQ658375), which contained 53 sequence differences
compared with the MNV-3 sequence of Hsu et al.
(2007) (Fig. 1a). These changes are the likely result of
additional passage of the virus in cell culture. The
pT7:MNV-3 construct was used to recover infectious
MNV-3 in cell culture as described previously (Chaudhry
et al., 2007). Briefly, BSR-T7 cells previously infected with
a recombinant fowlpox virus expressing recombinant T7
polymerase (FPV-T7) were transfected with pT7:MNV-3
or a similar wild-type MNV-1-containing construct
(pT7:MNV-1). Virus titres obtained at both 24 and
48 h post-transfection were noticeably lower for the
MNV-3 clone (2.16103and 6.36103TCID50ml21) than
forMNV-1 (2.16104and3.56104TCID50ml21)(Fig. 1b),
but recovery was reproducible and could also be obtained
using in vitro-transcribed and enzymically capped RNA
as described previously (Yunus et al., 2010) (data not
MNV-3 derived entirely from cDNA can be
propagated successively in RAW264.7 cells
MNV-3 recovered above, referred to as passage 0 (MNV-3
p0), was used to infect RAW264.7 cells at an approximate
m.o.i. of 0.001 TCID50 per cell. Ninety-six hours post-
infection, cytopathic effect was not apparent. Infectious
MNV-3 passage 1 (MNV-3 p1) was released by two
successive freeze–thaw cycles and the filtered lysate was
used to inoculate a new monolayer of RAW264.7 cells
(m.o.i. 0.1). MNV-3 p2 was recovered at 48 h post-
infection, associated with large degrees of cytopathic effect.
Consecutive passages of MNV-3 at m.o.i.s ranging from 0.1
to 0.3 resulted in an increase in the virus titre and in the
cytopathology in RAW264.7 cells during infection. Virus
yields (titres) by passage 6 (MNV-3 p6) were typically 15-
fold higher than at passage 2 (Fig. 1c). This was also
followed by an increase in the appearance of cytopathic
effect (not shown).
To determine whether increased virus yield was associated
with changes in the viral genome, viral RNA was extracted
from MNV-3 p2 and p6 stocks and their full genomes were
amplified by RT-PCR. Sequence analysis revealed only two
sequence changes in MNV-3 p6 relative to the cDNA clone
and in both cases a degree of heterogeneity was observed;
typical chromatogram signal intensities were in the range
60–80%/40–20% for the mutant/wild-type sequence. The
mutations identified were A2269G and C5957U, resulting
in the changes D50G and T301I in the viral NS4 and VP1
proteins, respectively. No mutations were found in the
MNV-3 p2 genome, suggesting that changes in MNV-3 p6
may reflect tissue-culture adaptation. We and others have
previously observed that, during repeated passage of MNV-
1 in RAW264.7 cells, similar adaptation occurs, with
changes also being observed in NS4 and VP1 (V11I and
K296E, respectively; Bailey et al., 2008; Wobus et al., 2004).
MNV-3 recovered by reverse genetics establishes
persistent infections in C57BL/6 mice
To investigate whether MNV-3 recovered in cell culture
from cDNA was able to establish a persistent infection in
immunocompetent mice, as shown previously for field
isolates of MNV-3 in ICR mice (Hsu et al., 2006), we
performed oral inoculations of 4–5-week-old C57BL/6
male mice. The maximum possible dose of virus was used
in this initial trial, namely 100 ml of either MNV-3 p2 or
MNV-3 p6 (1.06105and 1.56106TCID50, respectively).
C57BL/6 mice were chosen due to the ready availability of a
wide number of knockout and knock-in lines in this
genetic background. Inoculated animals showed no clinical
symptoms or differences in weight increase relative to
mock-infected or uninfected animals for the whole period
of surveillance (56 days) (Fig. 2).
To determine whether the cDNA-derived virus had estab-
lished a persistent infection, stool samples were collected
from the animals at days 28 and 56 post-infection, and
A. Arias and others
1434Journal of General Virology 93
analysed by both quantitative and semiquantitative RT-
PCR. Two different methodologies were used for further
validation of the levels of viral RNA and to overcome
limitations observed with the detection limits of each assay.
We detected genomic MNV RNA in the faeces of all the
inoculated animals at both days 28 and 56 post-infection
by semiquantitative RT-PCR, but not in faeces of mock-
infected animals (Fig. 2). Semiquantitative and quantitative
analyses were in agreementwith each other,andshowedthat
most animals excreted viral RNA at day 28 in the range of
105molecules (mg stool)21[corresponding to approx. 104
molecules of viral RNA (ml sample analysed)21] (Fig. 2). At
day 56, a decrease in viral RNA levels was observed when
compared with the same animals at day 28 [approx. 103–104
molecules (mg stool)21; P,0.05, two-way ANOVA], sug-
gesting that virus replication had been partially restricted.
Although not statistically significant, mean values for
animals infected with MNV-3 p2 tended to be higher than
in animals infected with MNV-3 p6. Sequence analysis of
MNV-3 VP1 in faeces of three different animals infected
with MNV-3 p6 (day 56) indicated that the dominant
sequence present at amino acid position 301 in VP1 was T.
This result might suggest that adaptive mutation T301I,
selected duringpassagein RAW264.7cells andpresent in the
passage 6 stock but absent after two passages, resulted in
decreased fitness in vivo and was outcompeted by wild-type
variants, detected as a minority in MNV-3 p6.
MNV-3 infection occurs at a low inoculating dose
and shows fast replication kinetics in mice
To investigate the infectivity of MNV-3 in an immuno-
competent background in vivo, we inoculated C57BL/6
mice with 10-fold-increasing doses of MNV-3 p2, ranging
from 10 to 104TCID50(Fig. 3). At 28 days post-infection,
the four groups of animals inoculated with virus displayed
789 10 1314 15 16 17
18 19 20 21 22 23 24
789 10 13 14 15 16 17 1819 20 2122 23 24
Time post-infection (days) Time post-infection (days)
Weight relative to day 0 (%)
vRNA molecules (mg stool)–1
Fig. 2. MNV-3 recovered by reverse genetics establishes persistent infections in mice. C57BL/6 mice, 4–5 weeks of age, were
inoculated with 100 ml sample containing 1.0?105TCID50MNV-3 p2 or 1.5?106TCID50MNV-3 p6. Controls included
uninfected and mock-infected animals. (a) No significant differences were observed in the weight gain of inoculated animals (#,
p2; h, p6) compared with controls (m, uninfected; &, mock-infected). (b, c) Viral RNA was extracted from stool pellets
collected (#, p2; h, p6; &, mock-infected) and was detected by quantitative (b) and semiquantitative (c) RT-PCR in all
infected animals, but not in mock-infected animals. Quantitative and semiquantitative PCRs were performed as described in the
text. Horizontal bars in (a) and (b) represent mean values. ”ve, Negative control; Ld, DNA ladder.
Persistent animal model for murine norovirus 3
detectable viral RNA by RT-PCR, whereas mock-infected
animals or animals inoculated with 105TCID50 UV-
inactivated MNV-3 were negative for MNV RNA (Fig. 3
and data not shown). Hence, the ID50(amount required
to infect 50% of the animals) of MNV-3 is likely to be
,10 TCID50. We carried out the quantification of viral
RNA in animals previously infected with 104
10 TCID50 by quantitative PCR, and compared them
with the values obtained previously for animals inocu-
lated with 105TCID50(Figs 2 and 3). The three groups of
animals displayed similar mean values, although animals
infected with 105TCID50 presented higher concentra-
tions on average (although not significant by statistical
analysis, one-way ANOVA test).
To determine whether the viral RNA detected in the faeces
correlated with the presence of infectious virus in the stool
pellets, we carried out TCID50 assays for the samples
analysed by quantitative PCR (Fig. 3c). We detected
infectivity in the stool pellets of 17 out of 18 animals. In
the positive samples, virus titres ranged from 1.26103to
1.96105TCID50(g stool sample)21. The calculated virus
titres per stool pellet (20–50 mg on average) ranged from
5.26101to 6.16103TCID50shed in every faecal sample
after 28 days post-infection. Interestingly, all animals
initially inoculated with 10 TCID50MNV-3 p2 (with the
exception of animal 118, which was negative for infectivity)
released .200 TCID50in a single pellet.
Given the low inoculating dose required to establish an
MNV-3 infection in vivo, we decided to investigate the
replication kinetics of MNV-3 after low-dose inoculation.
Mice were inoculated with either 10 or 102TCID50and the
kinetics of viral RNA shedding in the faeces of each animal
infected were determined by quantitative PCR. The data
indicated that MNV-3 infection occurs rapidly, with all
animals actively secreting viral RNA on day 3 (Fig. 4).
MNV-3 persists in the caecum and large intestine
To identify the major tissue(s) for MNV-3 replication and
persistence in C57BL/6 mice in vivo, we extracted various
organs previously identified to support MNV replication.
RNA was then isolated from the spleen, liver, mesenteric
lymph node (MLN), different sections of the small
intestine (duodenum/jejunum, proximal and distal ileum),
caecum and colon from infected animals at 28 days post-
inoculation. Strikingly, the highest levels of viral RNA were
detected in the colon followed by the caecum, with mean
values .105and .104MNV molecules (mg total RNA)21,
respectively (Fig. 5). Semiquantitative RT-PCR was also
used to confirm these observations (not shown). MNV
Day 28 post-infection
MNV RNA molecules (mg stool)–1
TCID50 (g stool)–1
Day 28 post-infection
101 102 103 104 105 106 113 114 115 116 117 118 149 150 151 152 153 154 107 108 109 110 111 112
Fig. 3. Low-dose inoculation of MNV-3 results in establishment of viral persistence. C57BL/6 mice 4–5 weeks of age were
inoculated with 100 ml sample containing 104, 103, 102or 10 TCID50MNV-3, 105TCID50UV-inactivated MNV-3, or filtered
cell-culture lysate (Mock). (a) Animals infected with 10 or 104TCID50MNV-3 showed detectable viral RNA in faeces by day 28
post-infection. Animals infected with 102or 103TCID50were also positive (not included in the figure). Animals mock-inoculated
with UV-inactivated MNV-3 or cell-culture lysate presented no detectable viral RNA. (b) Viral RNA levels in faeces collected at
28 days post-infection from animals mock-infected (&) or infected with 10 (e), 104(h) or 105(#) TCID50MNV-3 (from Fig.
2). (c) Titration by TCID50in RAW264.7 cells of infectious virus isolated from faecal samples of animals shown in (b). Horizontal
bars in (b) and (c) represent mean values.
A. Arias and others
1436Journal of General Virology 93
RNA was also detected in the MLN of some animals, but at
considerably lower levels than in tissues from the large
Persistent replication of MNV-3 in C57BL/6 mice
results in the selection of repeated mutations in
NS7 and VP2
To determine whether persistent replication of MNV-3 in
mice resulted in the selection of adaptive mutations in the
viral genome, we sequenced genomic RNA extracted from
the faeces of five different animals at day 28 post-infection
and from one animal at day 56 post-infection (Fig. 6). We
identified three mutations repeated in three or more
different animals: U3574C, C4179U and A6690G, which
resulted in amino acid changes V13A and L215F in the NS7
polymerase and T4A in the VP2 minor capsid protein. In
addition, only three other non-synonymous mutations
were identified in the analysis: A381T and T441I in VP1,
found in two clones, and T91A in NS3, found in one clone
Here, we describe the establishment of a persistent in-
fection in vivo with MNV-3 recovered from cDNA by
reverse genetics. To our knowledge, this is the first time
that a persistent infection of C57BL/6 mice with MNV
recovered from cDNA has been achieved. However, during
the course of this study, a reverse-genetics system for the
CR6 strain of MNV, shown previously to establish a
persistent infection (Thackray et al., 2007), has been
described. Recombinant virus derived from this system has
to date only been used to identify virulence determinants in
the VP1 protein within a STAT12/2model (Strong et al.,
The combination of an efficient reverse-genetics system for
MNV-3 and an animal model to study stable persistent
infections constitutes a significant step forward in the de-
biology and pathogenesis. Persistent infections in humans by
HuNoV have also been reported, although they mainly affect
immunocompromised individuals (Capizzi et al., 2011;
Ludwig et al., 2008). Nevertheless, recent data indicate that
asymptomaticnorovirus prevalenceisapproximately 12% in
the UK, although whether this represents subclinical acute
infections or long-term asymptomatic secretion of virus is
not known (Phillips et al., 2010). A recent report has shown
that MNV-3 infection in STAT12/2mice results in an acute
infection with typical signs of gastroenteritis (i.e. delayed
gastric emptying, changes to diarrhoeal symptoms) (Kahan
et al., 2011). This, combined with the fact that MNV-3 can
establish a persistent infection in C57BL/6 mice, the most
commonly used strain for the generation of knockout mice,
provides additional support for the use of MNV-3 for the
study of norovirus biology.
The observation that high viral RNA levels were detected in
caecum and colon at day 28 post-infection, but not in the
small intestine or other organs (MLN, spleen, liver), re-
presents one of the most significant findings of this study.
These data suggest that MNV-3 establishes a persistent
vRNA molecules (mg stool)–1
Time post-infection (days)
Fig. 4. MNV-3 shows fast kinetics of replication in mice. Groups of
six mice were infected with a low dose of MNV-3 [10 ($) or 102(h)
TCID50] and secretion of virus was monitored at days 1, 2, 3, 7 and
28 post-infection. MNV-3 shows fast replication kinetics, reaching
maximum levels of RNA molecules in stools by day 2 post-infection.
Establishment of infection is dose-dependent, with three of six
animals inoculated with 102TCID50, and only one of six animals
inoculated with 10 TCID50, being positive for virus shedding at
1 day post-inoculation. Horizontal bars represent mean values.
vRNA molecules (µg total RNA)–1
Fig. 5. MNV-3 persists in the caecum and the colon. High levels of
MNV RNA were detected in caecum and colon samples of animals
28 days post-inoculation. Lower amounts of viral RNA were
detected in MLN and small intestine of some animals. MNV RNA
levels in liver and spleen were below the detection limit [,4?102
viral RNA molecules (mg total RNA)”1]. The organs and tissues
collected included liver, spleen, MLN, different 1 inch sections of
the small intestine comprising dudodenum/jejunum (1–2 inches
from the stomach end), proximal ileum (3–4 inches from the
stomach end) and distal ileum (the last inch before the caecum),
caecum and colon. Horizontal bars represent mean values; the
dotted line represents the limit of detection.
Persistent animal model for murine norovirus 3
infection in the large intestine, allowing efficient virus
shedding in faeces over a prolonged time frame. The strong
association between ulcerative colitis, a colon disease, and
the presence of HuNoV during exacerbated disease (Khan
etal., 2009)supportstheidea that the colon mayalso play an
important role for HuNoV persistence. Two additional
studies have provided further evidence for the connection
between various colon disorders and active secretion of
HuNoV in faeces, specifically in children with persistent
diarrhoea and in patients with chronic diarrhoea associated
with leukaemia (Capizzietal.,2011;Vernacchio etal., 2006).
A recent study comparing MNV-1 and MNV-3 in vivo has
shown that the viruses replicate in different organs,
including the caecum and the colon, but they are rapidly
cleared, being undetectable by day 7 post-infection (Kahan
et al., 2011). Possible explanations for these apparently
conflicting data may be the sensitivity of the assays used and
differences in the experimental set-up, i.e. age and strain of
mice. It is also worth noting that, whilst the MNV-3 used
here was derived from cDNA, that used in the previous
study was a virus passed in tissue culture several times.
Interestingly, MNV persistent infections have also been
established in 7- to 15-week-old mice using the CR6 strain,
with viral RNA also being detected in colon 14 days post-
infection at levels lower than those observed in the ileum
and MLN (Cadwell et al., 2010). The results presented in
this study differ somewhat from these results, which may
be a reflection of the differing virus strain under study
(CR6 versus MNV-3) or the age of the animals used, i.e. 7–
15 weeks versus 4–5 weeks in our study. Alternatively,
MNV replication for longer periods (.14 days) may have
resulted in clearance from the ileum and MLN, but not
from the caecum and the colon.
Analysis of full MNV-3 genome sequences derived from
serial passage of MNV-3 in RAW264.7 cells and persis-
tent replication in mice resulted in marked differences in
genome evolution. Serial passage of MNV-3 in cell culture
selected for non-synonymous mutations in NS4 and VP1.
We have previously reported the selection of mutations in
NS4 and VP1 during MNV-1 cell-culture passage: K296E
in VP1 and V11I in NS4. K296E in MNV-1 VP1 was shown
to cause attenuation in STAT12/2mice (Bailey et al., 2008;
Strong et al., 2012). Interestingly, MNV-3 and MNV CR6,
both causative of persistent infections in wild-type mice,
encode E296 in VP1 instead of K. The substitution E296K
in MNV CR6 resulted in non-recoverable virus, which may
indicate that this mutation is not tolerated in the context of
the CR6 VP1 (Strong et al., 2012). Position 296 lies in close
proximity to position 301 in the structure of VP1, both at
the tip of the protruding P2 domain (Taube et al., 2010),
which we interestingly observed to be mutated (T301I)
during repeated passage of MNV-3 in RAW264.7 cells. Our
observations would suggest that, upon inoculation of
animals with a mixed population of viruses containing
both T and I at position 301, namely MNV-3 p6, only T at
position 301 in MNV-3 VP1 persists in vivo. Clearly,
further studies are warranted to examine the role of this
position in tissue-culture adaptation and the possible
associated fitness cost in vivo. In addition, adaptive
mutations in the picornavirus homologue of NS4 (3A)
have been linked to increased virulence in cell culture and/
or in vivo (Arias et al., 2010; Harris & Racaniello, 2005;
Nu ´n ˜ez et al., 2001), warranting further studies on the
precise function of the norovirus NS4 protein.
Persistent replication of MNV-3 in vivo did not select for
any of the substitutions found during cell-culture passage,
Fig. 6. Sequence analysis of viral RNA from stools at days 28 (animals 113, 122, 149, 152, 154) and 56 (animal 16, which
presented the highest viral RNA concentration at that time point). Empty and filled symbols represent synonymous and non-
synonymous substitutions, respectively. e/Xrepresent mutations not totally imposed in the consensus sequence (30–80%
relative to wild-type sequence based on chromatogram quantification); $ represents a totally imposed change.
A. Arias and others
1438 Journal of General Virology 93
but instead selected for changes in VP2 and NS7. The
mutations T4A in VP2, and V13A and L215F in NS7 were
found in six, four and three animals, respectively. Additional
mutations identified include A381T and T441I in VP1, and
T91A in NS3. The T4A substitution in VP2 was found in the
faeces of all animals analysed, but not during the serial
passage of MNV-3 in RAW264.7, suggesting that thischange
may be important in vivo but not relevant for cell-culture
replication. For the related feline calicivirus, the VP2 protein
is essential for virus replication and for the assembly of
infectious particles (Sosnovtsev et al., 2005). Recent studies
with HuNoV VP1 and VP2 have revealed co-evolution of
both proteins in a time-dependent manner, highlighting the
integral role of the VP2 protein in the norovirus life cycle
(Chan et al., 2012). Strikingly, we have found that alanine at
position 4 in VP2 is absolutely conserved in .30 natural
MNV isolates analysed. Nevertheless, the presence of threo-
nine at position 4 in VP2 had no effect on virus recovery by
reverse genetics or on virus replication in cell culture, being
stably maintained after multiple passages in RAW264.7 cells.
This highlights that the VP2 sequence may contribute to
virus fitness in vivo dueto selective pressures not observed in
Mutations V13A and L215F in the viral RNA polymerase
NS7 (Fig. 6) appear to affect polymerase surface residues,
distant from the catalytic site, in the palm and fingers
domains, respectively (Lee et al., 2011). Given their
accessibility, these changes may be affecting the interaction
of NS7 with potential viral or cellular factors, although this
hypothesis requires further investigation. Interestingly,
substitutions found in VP1 at lower frequency (A381T
and T441I) are predicted to lie within exposed residues of
flexible loops situated in the apical region of P2 and P1
domains, respectively, which could be indicative of
selection by neutralizing antibody response. In particular,
A381 is situated in loop E9-F9, identified previously as a
major immunodominant epitope with antibody-escape
mutants being mapped in position 386 (Lochridge &
Hardy, 2007; Taube et al., 2010).
In summary, we have described for the first time the
establishment of a persistent infection of C57BL/6 mice
with MNV-3 recovered by reverse genetics. MNV-3 derived
from cDNA persistently replicated in C57BL/6 mice for at
least 56 days and was associated with high viral loads in the
caecum and the colon. Preliminary data indicate that viral
RNA is shed by infected mice for .6 months, emphasizing
the ability of recovered MNV-3 to establish long-term
persistent infections in vivo (N. McFadden, A. Arias,
I. Goodfellow & P. Simmonds, unpublished results). This
model may open new possibilities to study norovirus
infections in vivo and different disorders associated with
Ethics. Studies with C57BL/6 mice were performed in the St Mary’s
CBS Unit of Imperial College London (PCD 70/2727) after ethical
review by the Imperial College Ethical Review Panel and subsequent
approval of the UK Home Office (PPL70/6838). All animal
procedures and care conformed strictly to the UK Home Office
Guidelines under The Animals (Scientific Procedures) Act 1986.
One-step construction of an infectious cDNA clone of MNV-3.
To enable the generation of a full-length cDNA clone of MNV-3, a
sample of MNV-3 provided by Robert Livingston (University of
Missouri, Columbia, MO, USA) was used to infect RAW264.7 cells at
an m.o.i. of 0.1 TCID50per cell (Hsu et al., 2007). RNA was extracted
from the infected cells 24 h post-infection. This sample collected
represented the viral sequence after three rounds of low-m.o.i.
infection in RAW264.7 cells. RNA was copied into cDNA using
SuperScript II (Invitrogen) and a reverse primer containing a 39 SpeI
site, a poly(A) of 27 nt and the last 36 nt of the MNV-3 genome.
Genomic MNV cDNA was fully amplified using KOD polymerase
(Merck), the primer used for reverse transcription and a 59 primer
containing a SpeI site, a truncated T7 RNA polymerase promoter and
the first 35 nt of the MNV genome. Gel-purified PCR product was
digested with SpeI and ligated into the pT7:MNV-1 backbone vector
digested with SpeI and NheI. The resulting pT7:MNV-3 contains 53
sequence changes (GenBank accession no. JQ658375) with respect to
the originally published MNV-3 sequence (Fig. 1) (Hsu et al., 2007).
Cells, infections and reverse-genetics recovery of MNV-3. Baby
hamster kidney cells expressing T7 RNA polymerase (BSR-T7)
obtained from Klaus Conzelmann (Ludwig-Maximilians-Universitat
Mu ¨nchen, Germany) (Buchholz et al., 1999) were cultured in
Dulbecco’s modified Eagle medium (DMEM) with 10% FCS,
100 U penicillin ml21and 100 mg streptomycin ml21. RAW264.7
cells, grown in DMEM with 10% FCS, 100 U penicillin ml21, 100 mg
streptomycin ml21and 10 mM HEPES pH 7.6, were used for the
titration (TCID50) and propagation of MNV-3.
For the recovery of infectious MNV-3, BSR-T7 cells were infected
with a helper fowlpox virus expressing recombinant T7 RNA
polymerase (FPV-T7) and transfected with pT7:MNV-3 as described
previously for MNV-1 (Chaudhry et al., 2007). Generated MNV-3
infectious particles were released from BSR-T7 cells by two
consecutive freeze–thaw cycles and supernatants were filtered. For
the amplification and generation of MNV-3 stocks, virus recovered
from BSR-T7 cells was inoculated on a monolayer of RAW264.7 cells
(m.o.i. 0.001) to obtain MNV-3 p1 (passage 1). Serial passage of
MNV-3 (passages 2–6) was carried out in RAW264.7 cells (m.o.i. 0.1–
0.3). Infected cells were then incubated at 37 uC until cytopathic effect
was apparent (typically 48 h) and then subjected to two consecutive
cycles of freezing and thawing and subsequent filtering to obtain
infectious particles. Virus titres were determined by TCID50assays in
RAW264.7 cells for 5 days followed by visual inspection.
Establishment of persistent MNV infections in vivo. Groups of
six C57BL/6 male mice of 4–5 weeks of age (Harlan or Charles River)
were inoculated by oral gavage with 100 ml sample containing varying
amounts of MNV-3 virus stocks. Mock-infected animals were
administered either filtered cell-culture lysates or 105TCID50 of
MNV-3 previously inactivated by UV exposure. For UV inactivation,
1.6 ml containing 26107TCID50of MNV-3 was cross-linked under
high-intensity UV light at 4 uC using a Spectrolinker XL-1500
TCID50titration of viral samples obtained from animal faeces.
Stool pellets collected from infected animals were placed on ice and
dispersed into PBS to reach a final concentration of 50 mg ml21.
Resuspended faeces werethen centrifuged at maximum speed (15000 g)
for 5 min and 100 ml supernatant was subjected to a second centrifu-
gation step to remove any traces of faecal debris. The double-purified
samples were titrated by TCID50assay in RAW264.7 cells.
Persistent animal model for murine norovirus 3
RNA extraction and RT-PCR-based detection of MNV-3 in
faeces and tissues. Viral RNA was extracted from 100 ml
supernatant of faeces dispersed into PBS (50 mg ml21) and from
an approximately 20 mg portion of each tissue following the
indications provided with the GenElute Mammalian Total RNA
Miniprep kit (Sigma-Aldrich). Different tissues/organs collected were
stored in RNAlater solution (Ambion) at 280 uC until RNA
extraction was performed. For semiquantitative detection of MNV-
3 RNA, one- or two-step RT-PCRs were performed using primers
spanning genomic residues 3395–3430 (forward) and 3770–3734
(reverse) (reference GenBank accession no. JQ658375), and Moloney
murine leukemia virus (M-MLV) reverse transcriptase and GoTaq
DNA polymerase (Promega). The resulting 376 bp product was then
resolved on a 2% agarose gel. To obtain an accurate determination of
the number of MNV-3 RNA molecules in faecal or tissue samples, we
carried out reverse transcription–quantitative PCR following proto-
cols described previously for the broad detection of MNV strains
(Kitajima et al., 2010). Reverse transcription of MNV cDNA was
performed using M-MLV reverse transcriptase (Promega) and a
primer complementary to genomic positions 5345–5380. MNV cDNA
was then quantified by quantitative PCR with primers spanning
residues 5028–5047 (sense) and 5177–5138 (antisense), and a
TaqMan FAM-TAMRA-labelled probe complementary to residues
5077–5062. Quantitative PCR determinations were carried out with
Precision 26 qPCR MasterMix (Primerdesign) in a ViiA7 Real-Time
PCR system apparatus (Applied Biosystems). In all the experiments, a
standard curve for MNV RNA with a known number of molecules
was carried out in parallel.
We would like to thank Professor Robert Livingston (University of
Missouri, Columbia, MO, USA) for supplying the MNV-3 strain used
here to prepare the plasmid harbouring the MNV-3 sequence. This
work has been funded by a Wellcome Trust Senior Fellowship, an Intra
European Fellowship given by the 7th Framework Program (Marie
Curie Actions), a Wellcome Trust Value In People Award, and support
from the Imperial College NIHR Biomedical Research Centre.
Arias, A., Perales, C., Escarmı ´s, C. & Domingo, E. (2010). Deletion
mutants of VPg reveal new cytopathology determinants in a
picornavirus. PLoS ONE 5, e10735.
Bailey, D., Thackray, L. B. & Goodfellow, I. G. (2008). A single amino
acid substitution in the murine norovirus capsid protein is sufficient
for attenuation in vivo. J Virol 82, 7725–7728.
Bailey, D., Karakasiliotis, I., Vashist, S., Chung, L. M., Rees, J.,
McFadden, N., Benson, A., Yarovinsky, F., Simmonds, P. &
Goodfellow, I. (2010). Functional analysis of RNA structures present
at the 39 extremity of the murine norovirus genome: the variable
polypyrimidine tract plays a role in viral virulence. J Virol 84, 2859–
Barron, E. L., Sosnovtsev, S. V., Bok, K., Prikhodko, V., Sandoval-
Jaime, C., Rhodes, C. R., Hasenkrug, K., Carmody, A. B., Ward, J. M. &
other authors (2011). Diversity of murine norovirus strains isolated
from asymptomatic mice of different genetic backgrounds within a
single U.S. research institute. PLoS ONE 6, e21435.
Buchholz, U. J., Finke, S. & Conzelmann, K. K. (1999). Generation of
bovine respiratory syncytial virus (BRSV) from cDNA: BRSV NS2 is
not essential for virus replication in tissue culture, and the human
RSV leader region acts as a functional BRSV genome promoter.
J Virol 73, 251–259.
Byrnes, A. P. & Griffin, D. E. (2000). Large-plaque mutants of Sindbis
virus show reduced binding to heparan sulfate, heightened viremia,
and slower clearance from the circulation. J Virol 74, 644–651.
Cadwell, K., Patel, K. K., Maloney, N. S., Liu, T. C., Ng, A. C., Storer,
C. E., Head, R. D., Xavier, R., Stappenbeck, T. S. & Virgin, H. W.
(2010). Virus-plus-susceptibility gene interaction determines Crohn’s
disease gene Atg16L1 phenotypes in intestine. Cell 141, 1135–1145.
Capizzi, T., Makari-Judson, G., Steingart, R. & Mertens, W. C. (2011).
Chronic diarrhea associated with persistent norovirus excretion in
patients with chronic lymphocytic leukemia: report of two cases.
BMC Infect Dis 11, 131.
CDC (2002). Outbreak of acute gastroenteritis associated with
Norwalk-like viruses among
Afghanistan, May 2002. MMWR Morb Mortal Wkly Rep 51, 477–479.
Chan, M. C., Lee, N., Ho, W. S., Law, C. O., Lau, T. C., Tsui, S. K. &
Sung, J. J. (2012). Covariation of major and minor viral capsid
proteins in norovirus genogroup II genotype 4 strains. J Virol 86,
Chaudhry, Y., Nayak, A., Bordeleau, M. E., Tanaka, J., Pelletier, J.,
Belsham, G. J., Roberts, L. O. & Goodfellow, I. G. (2006).
Caliciviruses differ in their functional requirements for eIF4F
components. J Biol Chem 281, 25315–25325.
Chaudhry, Y., Skinner, M. A. & Goodfellow, I. G. (2007). Recovery of
genetically defined murine norovirus in tissue culture by using a
fowlpox virus expressing T7 RNA polymerase. J Gen Virol 88, 2091–
Chen, S. Y., Tsai, C. N., Lai, M. W., Chen, C. Y., Lin, K. L., Lin, T. Y. &
Chiu, C. H. (2009). Norovirus infection as a cause of diarrhea-
associated benign infantile seizures. Clin Infect Dis 48, 849–855.
Daughenbaugh, K. F., Fraser, C. S., Hershey, J. W. & Hardy, M. E.
(2003). The genome-linked protein VPg of the Norwalk virus binds
eIF3, suggesting its role in translation initiation complex recruitment.
EMBO J 22, 2852–2859.
Daughenbaugh, K. F., Wobus, C. E. & Hardy, M. E. (2006). VPg of
murine norovirus binds translation initiation factors in infected cells.
Virol J 3, 33.
Goodfellow, I., Chaudhry, Y., Gioldasi, I., Gerondopoulos, A., Natoni,
A., Labrie, L., Laliberte ´, J. F. & Roberts, L. (2005). Calicivirus
translation initiation requires an interaction between VPg and eIF4E.
EMBO Rep 6, 968–972.
Harris, J. R. & Racaniello, V. R. (2005). Amino acid changes in
proteins 2B and 3A mediate rhinovirus type 39 growth in mouse cells.
J Virol 79, 5363–5373.
Harris, J. P., Edmunds, W. J., Pebody, R., Brown, D. W. & Lopman,
B. A. (2008). Deaths from norovirus among the elderly, England and
Wales. Emerg Infect Dis 14, 1546–1552.
Hsu, C. C., Riley, L. K., Wills, H. M. & Livingston, R. S. (2006).
Persistent infection with and serologic cross-reactivity of three novel
murine noroviruses. Comp Med 56, 247–251.
Hsu, C. C., Riley, L. K. & Livingston, R. S. (2007). Molecular charac-
terization of three novel murine noroviruses. Virus Genes 34, 147–155.
Ito, S., Takeshita, S., Nezu, A., Aihara, Y., Usuku, S., Noguchi, Y. &
Yokota, S. (2006). Norovirus-associated encephalopathy. Pediatr
Infect Dis J 25, 651–652.
Kahan, S. M., Liu, G., Reinhard, M. K., Hsu, C. C., Livingston, R. S. &
Karst, S. M. (2011). Comparative murine norovirus studies reveal a
lack of correlation between intestinal virus titers and enteric
pathology. Virology 421, 202–210.
Karst, S. M., Wobus, C. E., Lay, M., Davidson, J. & Virgin, H. W., IV
(2003). STAT1-dependent innate immunity to a Norwalk-like virus.
Science 299, 1575–1578.
A. Arias and others
1440 Journal of General Virology 93
Khan, R. R., Lawson, A. D., Minnich, L. L., Martin, K., Nasir, A., Download full-text
Emmett, M. K., Welch, C. A. & Udall, J. N., Jr (2009). Gastrointestinal
norovirus infection associated with exacerbation of inflammatory
bowel disease. J Pediatr Gastroenterol Nutr 48, 328–333.
Kitajima, M., Oka, T., Takagi, H., Tohya, Y., Katayama, H., Takeda, N.
& Katayama, K. (2010). Development and application of a broadly
reactive real-time reverse transcription-PCR assay for detection of
murine noroviruses. J Virol Methods 169, 269–273.
Koo, H. L., Ajami, N., Atmar, R. L. & DuPont, H. L. (2010). Noroviruses:
the leading cause of gastroenteritis worldwide. Discov Med 10, 61–70.
Lee, J. H., Alam, I., Han, K. R., Cho, S., Shin, S., Kang, S., Yang, J. M. &
Kim, K. H. (2011). Crystal structures of murine norovirus-1 RNA-
dependent RNA polymerase. J Gen Virol 92, 1607–1616.
Lencioni, K. C., Seamons, A., Treuting, P. M., Maggio-Price, L. &
Brabb, T. (2008). Murine norovirus: an intercurrent variable in a
mouse model of bacteria-induced inflammatory bowel disease. Comp
Med 58, 522–533.
Lochridge, V. P. & Hardy, M. E. (2007). A single-amino-acid
substitution in the P2 domain of VP1 of murine norovirus is
sufficient for escape from antibody neutralization. J Virol 81, 12316–
Lopman, B. A., Reacher, M. H., Vipond, I. B., Hill, D., Perry, C.,
Halladay, T., Brown, D. W., Edmunds, W. J. & Sarangi, J. (2004).
Epidemiology and cost of nosocomial gastroenteritis, Avon, England,
2002–2003. Emerg Infect Dis 10, 1827–1834.
Ludwig, A., Adams, O., Laws, H. J., Schroten, H. & Tenenbaum, T.
(2008). Quantitative detection of norovirus excretion in pediatric
patients with cancer and prolonged gastroenteritis and shedding of
norovirus. J Med Virol 80, 1461–1467.
McFadden, N., Bailey, D., Carrara, G., Benson, A., Chaudhry, Y.,
Shortland, A., Heeney, J., Yarovinsky, F., Simmonds, P. & other
authors (2011). Norovirus regulation of the innate immune response
and apoptosis occurs via the product of the alternative open reading
frame 4. PLoS Pathog 7, e1002413.
Mead, P. S., Slutsker, L., Dietz, V., McCaig, L. F., Bresee, J. S.,
Shapiro, C., Griffin, P. M. & Tauxe, R. V. (1999). Food-related illness
and death in the United States. Emerg Infect Dis 5, 607–625.
Monroe, S. S. (2011). Control and prevention of viral gastroenteritis.
Emerg Infect Dis 17, 1347–1348.
Mumphrey, S. M., Changotra, H., Moore, T. N., Heimann-Nichols,
E. R., Wobus, C. E., Reilly, M. J., Moghadamfalahi, M., Shukla, D. &
Karst, S. M. (2007). Murine norovirus 1 infection is associated with
histopathological changes in immunocompetent hosts, but clinical
disease is prevented by STAT1-dependent interferon responses. J Virol
Nu ´n ˜ez, J. I., Baranowski, E., Molina, N., Ruiz-Jarabo, C. M., Sa ´nchez,
C., Domingo, E. & Sobrino, F. (2001). A single amino acid
substitution in nonstructural protein 3A can mediate adaptation of
foot-and-mouth disease virus to the guinea pig. J Virol 75, 3977–3983.
Patel, M. M., Widdowson, M. A., Glass, R. I., Akazawa, K., Vinje ´, J. &
Parashar, U. D. (2008). Systematic literature review of role of
noroviruses in sporadic gastroenteritis. Emerg Infect Dis 14, 1224–
Phillips, G., Tam, C. C., Rodrigues, L. C. & Lopman, B. (2010).
Prevalence and characteristics of asymptomatic norovirus infection in
the community in England. Epidemiol Infect 138, 1454–1458.
Sosnovtsev, S. V., Belliot, G., Chang, K. O., Onwudiwe, O. & Green,
K. Y. (2005). Feline calicivirus VP2 is essential for the production of
infectious virions. J Virol 79, 4012–4024.
Sosnovtsev, S. V., Belliot, G., Chang, K. O., Prikhodko, V. G.,
Thackray, L. B., Wobus, C. E., Karst, S. M., Virgin, H. W. & Green, K. Y.
(2006). Cleavage map and proteolytic processing of the murine
norovirus nonstructural polyprotein in infected cells. J Virol 80,
Strong, D. W., Thackray, L. B., Smith, T. J. & Virgin, H. W. (2012).
Protruding domain of capsid protein is necessary and sufficient to
determine murine norovirus replication and pathogenesis in vivo.
J Virol 86, 2950–2958.
Taube, S., Rubin, J. R., Katpally, U., Smith, T. J., Kendall, A., Stuckey,
J. A. & Wobus, C. E. (2010). High-resolution X-ray structure and
functional analysis of the murine norovirus 1 capsid protein
protruding domain. J Virol 84, 5695–5705.
Thackray, L. B., Wobus, C. E., Chachu, K. A., Liu, B., Alegre, E. R.,
Henderson, K. S., Kelley, S. T. & Virgin, H. W., IV (2007). Murine
noroviruses comprising a single genogroup exhibit biological diversity
despite limited sequence divergence. J Virol 81, 10460–10473.
van Asten, L., Siebenga, J., van den Wijngaard, C., Verheij, R., van
Vliet, H., Kretzschmar, M., Boshuizen, H., van Pelt, W. & Koopmans,
M. (2011). Unspecified gastroenteritis illness and deaths in the elderly
associated with norovirus epidemics. Epidemiology 22, 336–343.
Vernacchio, L., Vezina, R. M., Mitchell, A. A., Lesko, S. M., Plaut, A. G.
& Acheson, D. W. (2006). Characteristics of persistent diarrhea in a
community-based cohort ofyoung
Gastroenterol Nutr 43, 52–58.
US children.J Pediatr
Walker, S. C., Avis, J. M. & Conn, G. L. (2003). General plasmids for
producing RNA in vitro transcripts with homogeneous ends. Nucleic
Acids Res 31, e82.
Ward, V. K., McCormick, C. J., Clarke, I. N., Salim, O., Wobus, C. E.,
Thackray, L. B., Virgin, H. W., IV & Lambden, P. R. (2007). Recovery of
infectious murine norovirus using pol II-driven expression of full-
length cDNA. Proc Natl Acad Sci U S A 104, 11050–11055.
Wobus, C. E., Karst, S. M., Thackray, L. B., Chang, K. O., Sosnovtsev,
S. V., Belliot, G., Krug, A., Mackenzie, J. M., Green, K. Y. & Virgin, H.
W. (2004). Replication of norovirus in cell culture reveals a tropism
for dendritic cells and macrophages. PLoS Biol 2, e432.
Wobus, C. E., Thackray, L. B. & Virgin, H. W., IV (2006). Murine
norovirus: a model system to study norovirus biology and
pathogenesis. J Virol 80, 5104–5112.
Yunus, M. A., Chung, L. M., Chaudhry, Y., Bailey, D. & Goodfellow, I.
(2010). Development of an optimized RNA-based murine norovirus
reverse genetics system. J Virol Methods 169, 112–118.
Persistent animal model for murine norovirus 3