JOURNAL OF VIROLOGY, Sept. 2004, p. 10156–10165
Copyright © 2004, American Society for Microbiology. All Rights Reserved.
Vol. 78, No. 18
Ceacam1a?/?Mice Are Completely Resistant to Infection by Murine
Coronavirus Mouse Hepatitis Virus A59
Erin Hemmila,1Claire Turbide,2Melanie Olson,2Serge Jothy,3Kathryn V. Holmes,1† and
Department of Microbiology, University of Colorado Health Sciences Center, Denver, Colorado,1and McGill Cancer
Centre2and Departments of Biochemistry, Medicine, and Oncology,4McGill University, Montreal, Quebec,
and Department of Laboratory Medicine and Pathobiology, St-Michael’s Hospital,
Received 29 December 2003/Accepted 10 May 2004
CEACAM1a glycoproteins are members of the immunoglobulin (Ig) superfamily and the carcinoembryonic
antigen family. Isoforms expressing either two or four alternatively spliced Ig-like domains in mice have been
found in a number of epithelial, endothelial, or hematopoietic tissues. CEACAM1a functions as an intercel-
lular adhesion molecule, an angiogenic factor, and a tumor cell growth inhibitor. Moreover, the mouse and
human CEACAM1a proteins are targets of viral or bacterial pathogens, respectively, including the murine
coronavirus mouse hepatitis virus (MHV), Haemophilus influenzae, Neisseria gonorrhoeae, and Neisseria men-
ingitidis, as well as Moraxella catarrhalis in humans. We have shown that targeted disruption of the Ceacam1a
(MHVR) gene resulting in a partial ablation of the protein in mice (p/p mice) led to reduced susceptibility to
MHV-A59 infection of the modified mice in the BALB/c background. We have now engineered and produced a
Ceacam1a?/?mouse that exhibits complete ablation of the CEACAM1a protein in every tissue where it is
normally expressed. We report that 3-week-old Ceacam1a?/?mice in the C57BL/6 genetic background are fully
resistant to MHV-A59 infection by both intranasal and intracerebral routes. Whereas virus-inoculated wild-
type ?/? C57BL/6 mice showed profound liver damage and spinal cord demyelination under these conditions,
Ceacam1a?/?mice displayed normal livers and spinal cords. Virus was recovered from liver and spinal cord
tissues of ?/? mice but not of ?/? mice. These results indicate that CEACAM1a is the sole receptor for
MHV-A59 in both liver and brain and that its deletion from the mouse renders the mouse completely resistant
to infection by this virus.
The carcinoembryonic antigen (CEA) family comprises a
large number of immunoglobulin (Ig)-like genes clustered on
human chromosome 19q13.2 (40). The Ceacam1a gene is
thought to represent the ancestor of this family, as it is well
conserved in its gene structure, alternatively spliced isoforms,
its expression patterns, and its functions in rodents and hu-
mans (15). The nomenclature of this large family has recently
been redefined and standardized (3). The mouse genome con-
tains two Ceacam-like genes, Ceacam1 and Ceacam2 (formerly
known as Bgp1 and Bgp2) (28, 29). Although the gene struc-
tures are highly homologous, the proteins they encode exhibit
a very different pattern of expression, with CEACAM1a pro-
tein most abundant in liver, intestine, kidney, and hematopoi-
etic cells (4), whereas CEACAM2 is present in kidney and
pancreas (16, 34). The mouse CEACAM1a isoforms contain
either two or four extracellular alternatively spliced Ig-like
domains that are tethered to the cell membrane via their trans-
membrane domain and display two different intracytoplasmic
domains designated short (S; 10 amino acids [aa]) or long (L;
73 aa). The long cytoplasmic domain is produced by insertion
of the 53-bp exon 7 of the Ceacam1a gene that switches the
open reading frame of the encoded transcript. This results in
an extended cytoplasmic tail by translation of 63 aa, containing
inhibitory tyrosine-based immunoreceptor motifs (ITIM) that
are well conserved across species (24, 33).
CEACAM1a expression in tissues of mice and humans is
widespread, although not ubiquitous. CEACAM1a proteins
are abundantly expressed at the surface of epithelial cells of
the gastrointestinal and respiratory tracts, particularly in the
bile canaliculi, the intestine, the proximal tubules of the kid-
ney, and the lung (12, 25, 30, 31). These glycoproteins are also
found on small vascular endothelial cells and in hematopoietic
cells (such as activated T cells, B lymphocytes, neutrophils,
macrophages, monocytes, platelets, and thymic stromal cells)
(6, 10, 12, 14, 26, 27). CEACAM1a isoforms are also displayed
on epithelial cells of reproductive tissues such as the uterus,
the breast, and the prostate (18, 19, 38). Moreover, the pro-
teins are expressed at low levels in the glial cells of the nervous
system (12, 35). In these tissues, CEACAM1a exhibits a num-
ber of functions. Briefly, CEACAM1a acts as a cell adhesion
molecule, an angiogenic factor, a tumor suppressor, and a
signal regulatory protein (3).
Furthermore, human CEACAM1a is a receptor for a num-
ber of bacterial pathogens, such as Escherichia coli, Salmonella
enterica serovar Typhimurium, Neisseria gonorrhoeae, Neisseria
meningitidis, and Haemophilus influenzae, as well as Moraxella
catarrhalis (13, 17, 23, 42, 43). Likewise, murine CEACAM1a
proteins serve as receptors for a viral pathogen, murine coro-
navirus mouse hepatitis virus (MHV) (8). Two Ceacam1 alleles
* Corresponding author. Mailing address: McGill Cancer Centre,
McGill University, McIntyre Medical Sciences Building, 3655 Prome-
nade Sir-William-Osler, Montreal, Quebec, Canada H3G 1Y6. Phone:
(514) 398-3541. Fax: (514) 398-6769. E-mail: nicole.beauchemin
† K.V.H. and N.B. contributed equally to this work.
in various mouse strains have been identified. The Ceacam1a
allele confers susceptibility to MHV infection and is present in
most inbred mouse strains, as well as in outbred mice (8).
Adult SJL inbred mice carry the Ceacam1b allele and are fully
resistant to MHV infection (2, 21, 36). These two alleles differ
in 27 of the 108 aa of the first Ig domain of CEACAM1 (7).
Mutational analyses showed that the virus binds between aa 34
to 52 in the N-terminal domain of murine CEACAM1a (9, 32,
44). The crystal structure of soluble murine CEACAM1a do-
main 1 linked to domain 4 showed that the C-C? loop (aa 35 to
44) has a unique stable convoluted conformation with a pro-
jecting Ile41 that is postulated to be important for binding to
the viral spike glycoprotein (39). Binding of the spike glyco-
protein to CEACAM1a at 37°C induces conformational changes
in the spike protein that probably lead to fusion of the viral
envelope with the host cell membrane and virus infection (41,
We recently developed a mouse strain with a partial ablation
of the CEACAM1a proteins (labeled p/p mice). In this
BALB/c mouse model, the isoforms with four Ig domains were
reduced in expression by 90 to 95%, whereas expression of
those with two Ig domains was slightly increased. Upon intra-
nasal (i.n.) inoculation of these mice with MHV-A59, the p/p
mice did not show obvious clinical signs of infection. They had
significantly fewer and smaller liver lesions than their wild-type
littermate controls. Therefore, these genetically altered p/p
mice exhibited decreased susceptibility to MHV-A59 infection
We have now generated two mouse strains (2D2 and 11H11)
on a C57BL/6 background that have a complete ablation of the
CEACAM1a proteins (the strains are designated ?/?).
Ceacam1a?/?mice were inoculated with MHV-A59 by i.n. and
intracerebral (i.c.) routes, and histopathology, titers of infec-
tious virus, and expression of viral RNA in liver and brain were
compared with those of wild-type ?/? mice. In addition, we
have assessed the effects of i.c. inoculation of p/p BALB/c mice
with MHV-A59. These experiments showed that the virus
caused demyelination in the spinal cords of p/p mice without
clinical signs but that the demyelinated lesions were smaller
than thosein wild-type
Ceacam1a?/?mice were fully resistant to MHV infection and
had no virus replication or lesions in liver, brain, or spinal cord.
These data show that CEACAM1a is the sole receptor for
MHV-A59 in C57BL/6 mice.
MATERIALS AND METHODS
Generation of the Ceacam1a?/?mouse lines. Details of the procedures in-
volved in the generation of the Ceacam1a?/?mouse lines and their primary
analyses and phenotypes will be presented in detail in another manuscript (T
Dai et al., unpublished data). Briefly, a targeting vector was prepared in which
the first two exons of the Ceacam1a gene were replaced by a pTK-neorcassette,
thereby eliminating the exon coding for the initiator ATG codon as well as the
second exon containing the viral binding domain (5). This vector was electropo-
rated into R1 embryonic stem (ES) cells that were subjected to G418 analog
selection. Two ES cell lines (designated 2D2 and 11H11) positive for the inte-
gration event were verified for genomic locus integrity and microinjected into
C57BL/6 mouse blastocysts. Chimeric males were mated with C57BL/6 female
mice, and heterozygous and homozygous Ceacam1a mice were derived (the
mouse lines were designated 2D2 and 11H11). Experiments were performed in
the second and third backcrosses of both the 2D2 and 11H11 Ceacam1a?/?mice.
Care of the mice was according to the standards defined by the Canadian Council
on Animal Care.
Genotyping. Genotyping was performed with ?1 cm of tails clipped from
3-week-old pups; genomic DNA was prepared with a QIAamp DNA kit (QIA-
GEN). Approximately 5 ?g of genomic DNA was cleaved with EcoRI restriction
endonuclease and separated on 0.75% agarose gels. The DNA was transferred to
GeneScreen Plus membranes (NEN-Life Science Products, Boston, Mass.) and
hybridized at 42°C for 18 h with 2 ? 106to 4 ? 106dpm of random-primed
[?-32P]dATP-labeled restriction fragments (29). A 93-bp BamHI-HindIII frag-
ment cleaved from within the Ceacam1a promoter in a region located outside of
the targeting vector was used as a probe. Membranes were washed at a final
stringency of 65°C in a solution of 0.1? SSC (1? SSC is 0.15 M NaCl plus 0.015
M sodium citrate) and 0.1% sodium dodecyl sulfate (SDS). Alternatively, the
mice were genotyped by PCR amplification of their genomic DNA in a final
volume of 15 ?l containing a 3? dilution of Vent polymerase buffer supplied by
the manufacturer, 500 ?M of deoxynucleoside triphosphates, 5 ng of the
Ceacam1a-specific oligonucleotides (PN8, 5?CTGCCCCTGGCGCTTGGA;
PN5, 5?TACATGAAATCGCACAGTCGC)/?l, 5 ng of the neor-specific oligo-
nucleotides (neoforward, 5?CGGTGCCCTGAATGAACTGC; neoreverse, 5?G
CCGCCAAGCTCTTCAGCAA)/?l, and 0.4 U of Vent polymerase. Amplifica-
tions proceeded through 30 cycles of 20 s of denaturation at 94°C, 30 s of binding
at 57°C, and 30 s of elongation at 72°C. The wild-type ?/? mice exhibited a
Ceacam1a fragment of 250 bp upon agarose gel electrophoresis, whereas the
homozygous ?/? mice had a 550-bp neo fragment and the heterozygous ?/?
mice showed both fragments.
Sampling and preparation of tissues. The mice were anesthetized with Avertin
(Aldrich Chemical Company, Milwaukee, Wis.) and then sacrificed by either
cervical dislocation or exsanguination. Tissues were removed, washed in phos-
phate-buffered saline (PBS), and either snap-frozen on dry ice or liquid nitrogen
for subsequent DNA, RNA, protein, or viral titer analyses. Some tissues were
washed and fixed in 3.7% paraformaldehyde–PBS or 10% phosphate-buffered
formalin and processed for immunohistochemistry.
RNA preparation and Northern analysis. Mouse tissues were retrieved and
snap-frozen on dry ice. The tissues were then powdered with a mortar and pestle
kept at ?80°C, and the RNA was extracted with materials provided in the
RNAqueous kit (Ambion) following the manufacturer’s recommendation. Five
micrograms of total RNA was subjected to electrophoresis on formaldehyde-
agarose gels and transferred to Hybond N? (Amersham). The membrane was
hybridized with a full-length32P-labeled Ceacam1a cDNA for 18 h at 42°C and
washed in a solution of 0.1? SSC plus 0.1% SDS at 65°C. Membranes were
exposed to X-ray film for 18 to 96 h.
Antibodies (Abs), immunoblotting, and immunoprecipitations. Fresh tissues
were excised from 2- to 6-month-old Ceacam1a?/?, Ceacam1a?/?, or
Ceacam1a?/?mice, snap-frozen on dry ice, and powdered with a mortar and
pestle. The powder was resuspended in 500 to 1,000 ?l of lysis buffer (20).
Proteins were separated on SDS–8% polyacrylamide gel electrophoresis gels and
transferred to Immobilon membranes (Millipore, Nepean, Ontario, Canada).
Expression of the CEACAM1a isoforms was detected by immunoblotting 75 to
200 ?g of total proteins with the anti-CEACAM1a-specific rabbit polyclonal Ab
231 or 2456. Immune complexes were visualized with an ECL detection system
(Amersham Pharmacia Biotech, Baie d’Urfe ´, Quebec, Canada). The control
used in the experiments was a cell lysate from CEACAM1a-transfected NIH 3T3
cells (data not shown).
Histological analyses. Fixed tissues were dehydrated in ethanol and paraffin
embedded. Tissue sections were counterstained with hematoxylin and eosin
according to standard histological procedures.
MHV preparations and i.n. and i.c. inoculation of mice. The MHV-A59 virus
strain used in these experiments was propagated in the spontaneously trans-
formed 17 Cl 1 line of BALB/c 3T3 cells as previously described (11). The
supernatant medium was collected 24 h after inoculation, centrifuged to remove
cellular debris, aliquoted, quickly frozen, and stored at ?80°C. Titers of infec-
tious virus were determined by plaque assay of 17 Cl 1 cells (11). Three-week-old
Ceacam1a?/?and Ceacam1a?/?mice were inoculated i.n. with 10 ?l of virus
containing 106to 108PFU in Dulbecco’s PBS. To obtain concentrated virus for
the high-titer inoculum, virions were purified and concentrated by sucrose den-
sity ultracentrifugation as previously described (37). Control, uninfected ?/? or
?/? mice were sham inoculated i.n. with 10 ?l of PBS. The mice were observed
daily for clinical signs of illness, such as lethargy, ruffled fur, hunched posture, or
paralysis. Mice anesthetized intraperitoneally with Avertin were inoculated i.c.
with 103to 106PFU of the MHV-A59 virus in 10 ?l with a 27-gauge needle and
a 1-ml syringe fitted to a Tridak Stepper repetitive delivery device. Samples were
evaluated in the same fashion as those from the i.n. inoculated mice.
Analyses of tissues of inoculated mice. Mice were sacrificed at intervals of 4,
7, 14, 30, 45, and 60 days postinoculation (dpi). Serum samples were collected for
evaluation of anti-virus Abs. The livers, brains, and spinal cords were processed
VOL. 78, 2004MHV RESISTANCE OF Ceacam1a?/?MICE10157
to determine the yield of infectious virus and to study histopathology. To quan-
titate the infectious virus in the liver and the brain, portions of the liver or brain
removed at necropsy were rinsed in PBS, weighed, homogenized in Dulbecco’s
modified Eagle medium with 10% fetal bovine serum, and rapidly frozen and
thawed at 37°C three times. Cell debris was removed by centrifugation. The virus
titer per gram of liver or brain in the supernatant was determined by plaque assay
of 17 Cl 1 cells as described previously (11). Tissues were fixed in neutral buffered
formalin or 4% paraformaldehyde, embedded in paraffin, sectioned, and stained
with hematoxylin and eosin. Sections were examined by light microscopy, and the
numbers and sizes of lesions in comparable areas of the liver and brain sections
Reverse transcription-PCR (RT-PCR) analyses of viral RNA in brain and
liver. To detect viral RNA in infected tissues, RNA was isolated following
Invitrogen’s TRIzol reagent protocol. Briefly, approximately 100 mg of infected
mouse tissue was homogenized and solubilized in 1 ml of TRIzol at 20°C for 5
min. After chloroform extraction, RNA was precipitated from the top aqueous
phase. RNA, resuspended in H2O, was used as a template for RT with AMV
reverse transcriptase (Promega, Madison, Wis.). PCR to detect viral RNA was
performed with Taq DNA polymerase (Promega). Primers to amplify the nu-
cleocapsid and leader region of the coronavirus MHV-A59 mRNA for the E1
glycoprotein genome were designed according to sequences from the National
Center for Biotechnology Information (accession number X00509). First rounds
of PCR were performed with the forward primer 5?GTACGTACCCTCTCAA
CTCT and the reverse primer 5?CCCATCAGGTGTTTTAAAAG. The hemin-
ested PCRs were performed with the forward primer 5?TACATGAAATCGCA
CAGTCGC. Some samples were positive in the first rounds of PCR, and other
samples required heminested PCR to detect viral RNA. Both positive- and
negative-control samples were included in this assay.
Detection of anti-virus Abs. Mouse sera were tested for anti-virus Abs with an
enzyme-linked immunosorbent assay (ELISA) in Immulon-HP 96-well microti-
ter plates coated with 105PFU per well of purified MHV-A59 diluted in 50 ?l of
sodium carbonate buffer (pH 9.5). Plates for the ELISA were blocked overnight
in B3 buffer (150 mM NaCl, 50 mM Tris, 0.8 mM EDTA, 0.05% Tween [vol/vol],
0.01% bovine serum albumin [wt/vol] [pH 7.4]) plus 5% bovine serum albumin.
Anti-virus Ab titers in mouse serum in B3 buffer were assayed with anti-mouse
horseradish peroxidase-labeled anti-mouse Ig Ab and 2,2 N?-azinobis(3-ethyl-
benzthiazolinesulfonic acid) (ABTS) substrate (Kirkegaard and Perry Laborato-
ries, Gaithersburg, Md.). Absorbance was measured at 405 nm and plotted versus
Generation of the Ceacam1a?/?mice. The strategy leading
to complete abrogation of CEACAM1a expression in mice was
based on the removal of the first two exons of the Ceacam1a
gene (Fig. 1A and B). The initiator ATG codon is positioned
in the first exon, and many functions associated with the
CEACAM1a protein depend on the presence of the first Ig-
like domain encompassed within the second exon. For this
purpose, an Xba1-Xho1 restriction fragment carrying these
two exons was removed from the gene, and a cassette with the
TK (thymidine kinase) promoter and the neorgene was in-
serted (Fig. 1B). The targeting vector was electroporated into
mouse R1 ES stem cells, and chimeric mice were generated by
microinjection of two ES cell lines (2D2 and 11H11) into
C57BL/6 mouse blastocysts. Eight chimeric male mice were
obtained, four of which transmitted the Ceacam1a?/?-targeted
allele through the germ line. The heterozygous Ceacam1a?/?
progeny mice were mated to produce homozygous (?/?) mice.
The frequency of germ line transmission was calculated to be
22% on varied backgrounds (C57BL/6, BALB/c, and 129Sv).
Mating of heterozygous mice produced expected Mendelian
ratios of Ceacam1a?/?offspring (?/?, 1.0; ?/?, 1.8; ?/?,
0.9). CEACAM1a is expressed in ovary and prostate, yet its
ablation did not alter the sex ratio of the progeny (males, 52%;
Abrogation of CEACAM1a expression in Ceacam1a-targeted
mice. The complete abrogation of CEACAM1a expression was
verified by Northern and Western blotting. Total RNA was
prepared from colon and liver of several mice from each litter
and analyzed with formaldehyde-agarose gels. Northern blots
were produced and subjected to hybridization with the32P-
labeled full-length Ceacam1a cDNA (24). The wild-type ?/?
and heterozygous ?/? mice produced a 4-kb RNA fragment
corresponding to the Ceacam1a transcript. The intensity of the
RNA fragment in the ?/? mice was approximately half of that
in the control RNA produced from wild-type mice. No
Ceacam1a transcript was revealed in the homozygous ?/?
mice even after prolonged exposure of the membranes, indi-
cating that the gene inactivation strategy completely abrogated
Ceacam1a transcription (Fig. 1C). We then examined the ex-
pression of the CEACAM1a protein isoforms in mouse colon
and liver tissues by immunoblotting total proteins from several
different mice with anti-CEACAM1a polyclonal Abs (Fig. 1D).
In these and other tissues (data not shown), expression of all
CEACAM1a isoforms in the homozygous ?/? mice was com-
pletely eliminated relative to expression in the wild-type ?/?
or heterozygous ?/? mice (Fig. 1D). Actin protein levels in
these tissues were constant (Fig. 1E).
Absence of CEACAM1a expression in tissues of Ceacam1a-
deficient mice. By routine histology, no histological differences
were noted in the colon, small intestine, liver, kidney, prostate,
ovaries, uterus, brain, lungs, heart, and spleen of ?/? mice
compared to those of control ?/? mice (Fig. 2 and data not
shown). Paraffin-embedded tissue sections were immuno-
stained with anti-CEACAM1a polyclonal (Ab 2456) and
monoclonal (MAb-CC1) (data not shown) Abs. When immu-
nostained with an anti-CEACAM1a polyclonal Ab, tissues
from wild-type ?/? animals exhibited strong expression on the
luminal membrane of surface and crypt cells of the colon (Fig.
2a and b). Bile canaliculi of the liver and proximal tubules of
the kidney were also strongly positive for CEACAM1a in ?/?
mice (Fig. 2c and d). In contrast, in ?/? mice, colonic (Fig. 2e)
or intestinal (Fig. 2f) epithelial cells revealed no staining of the
crypts, even after a longer development time. This finding was
repeated for hepatocytes (Fig. 2h) and the collecting tubules in
the kidney (Fig. 2g) of ?/? mice. These tissues were also not
immunostained with anti-CEACAM1a polyclonal Abs. Other
tissues that normally express CEACAM1a (small intestine,
endometrium, ovary, prostate, stomach, spleen, thymus, and
lung) also displayed no immunostaining in the ?/? mice (data
General health status of the offspring. The Ceacam1a-tar-
geted mice were viable and healthy under a pathogen-free
environment. We have maintained a sizeable colony (approx-
imately 350 individuals) of targeted mice for 1 year on the
BALB/c, 129Sv, and C57BL/6 backgrounds and have not no-
ticed any reduction in fertility, bone or cartilage abnormalities,
tumors, or abnormal behavior. However, these mice do exhibit
distinct phenotypes relative to liver insulin resistance and
clearance (Dai et al., unpublished) and in in vivo T-cell phys-
iology that will be reported elsewhere (R. Atallah et al., un-
Results of i.n. inoculation of Ceacam1a?/?mice with MHV-
A59. To define whether the Ceacam1a-targeted mice were
susceptible or resistant to MHV infection, we subjected wild-
10158 HEMMILA ET AL.J. VIROL.
type ?/? and ?/? mice from both the 2D2 and 11H11 lines to
infection with the MHV-A59 virus. Mouse litters were gener-
ated by crossing Ceacam1a?/?siblings, crossing ?/? mice with
?/? mice, or mating ?/? mice. Experiments were performed
with the second and third backcrosses of both lines of
Ceacam1a?/?mice. Genotyping was performed as described
above on DNA prepared from mouse tails. Control mice were
sham –inoculated, and both wild-type ?/? and ?/? mice were
inoculated i.n. with 10 ?l of virus containing 106PFU of MHV-
A59 (Table 1). The mice were examined every day for clinical
signs of disease and, if warranted, were sacrificed immediately.
Groups of ?/? and ?/? mice were sacrificed at 4, 14, 30, 45,
and 60 dpi. Blood was collected to test for Abs to viral antigens
(serocoversion), and liver and spinal cord tissues were pro-
cessed for pathology. Liver and brain RNA was tested for the
presence of viral RNA that is indicative of viral infection.
At 4 and 14 dpi, the health of the ?/? mice was compro-
mised, as they showed many of the clinical symptoms described
in Materials and Methods, whereas the ?/? mice showed no
signs of illness. Ultimately, five infected ?/? animals survived
and recovered from the infection. Mice sacrificed between 30
and 60 dpi did not demonstrate overt clinical symptoms. As
seen in the results shown in Table 1, infected ?/? mice sacri-
ficed between 14 and 45 dpi generally exhibited high anti-virus
serum Ab titers. Conversely, only two of the Ceacam1a?/?
mice produced an Ab response of relatively low titer to viral
antigens. Only one of four ?/? mice had seroconverted at 14
dpi, none had mounted an immune response at 30 dpi or 45
dpi, and just one of five ?/? mice tested weakly positive at 60
dpi. Liver histology was examined for the presence of viral
lesions, and a grading system was established that took into
consideration the numbers and sizes of the lesions. At early
FIG. 1. Northern and Western analyses of Ceacam1a?/?mice. (A) The Ceacam1a gene comprises nine exons (numbered boxes) preceded by
the Ceacam1a promoter (Prom.). Exon 1 contains the initiator ATG codon at the end of the 5? untranslated region (5?UT) and the beginning of
the half-leader (L) sequence. The second exon encompasses the second half-leader sequence and the first Ig domain (D1). Exons 3, 4, and 5 encode
three constant-type Ig domains (D2, D3, and D4). Exon 6 contains the transmembrane domain (TM) and part of the cytoplasmic domain (C), the
rest being distributed in exons 7, 8, and 9. Two different stop codons [TGA(S)] and [TGS(L)] terminate the translation of either the CEACAM1a-S
or CEACAM1a-L isoforms. (B) A targeting vector was engineered whereby a TK-neorselection cassette was inserted into the XbaI site at the
beginning of the 5?UT and an XhoI site located in intron 2. The selection cassette removed the first two exons as well as the initiator ATG codon.
It was flanked on the 5? side by a 0.9-kb Ceacam1a promoter fragment and on the 3? side by a 4.1-kb fragment containing exons 3, 4, and 5.
(C) Northern analyses of colon and liver RNA from mouse progeny. Total RNA was prepared from colon and liver of several mice from each litter,
and 5 ?g of RNA was separated on formaldehyde-agarose gels. Northern blots were produced and subjected to hybridization with the32P-labeled
full-length Ceacam1a cDNA. Samples from wild-type ?/? and heterozygous ?/? mice produced a 4-kb RNA fragment corresponding to the
Ceacam1a transcript. The intensity of the RNA fragment in the ?/? mice was approximately half that of the control RNA from wild-type ?/?
mice. No Ceacam1a transcript was revealed in the homozygous ?/? mice even after prolonged exposure of the membranes. CT, hybridization of
total RNA prepared from a BALB/c mouse colon. (D) Tissues were excised from 2-month-old Ceacam1a?/?, ?/?, or ?/? mice, snap-frozen on
dry ice, and powdered with a mortar and pestle. The powder was resuspended in 500 ?l of lysis buffer. Samples of 200 ?g of proteins were separated
on SDS–8% PAGE gels and transferred to Immobilon membranes. Expression of the CEACAM1a isoforms was detected by immunoblotting of
total proteins with the anti-CEACAM1a-specific rabbit polyclonal Ab 2456. Immune complexes were visualized with an ECL detection system. 4D,
CEACAM1a isoforms containing four Ig domains; 2D, CEACAM1a isoforms with two Ig domains. The band appearing at approximately 97 kDa
is a degradation product. (E) The membrane shown in panel D was cleaned and reblotted with an anti-actin Ab.
VOL. 78, 2004MHV RESISTANCE OF Ceacam1a?/?MICE10159
time points after infection (4 and 14 dpi) (Table 1), all ?/?
mice exhibited liver pathology. Indeed, ?/? mice sacrificed at
4 dpi showed lesions involving from 10% (liver pathology
grade, 2?) up to more than 90% (4?) of cells (Fig. 3B and C,
row I). Some ?/? mice sacrificed at 14 dpi had resolving
lesions affecting up to 10% (1?) of cells. After the ?/? mice
recovered from signs of infection by the 30- to 60-dpi period,
the liver lesions were no longer detectable. None of the ?/?
FIG. 2. CEACAM1a immunostaining of tissue sections from wild-type ?/? and Ceacam1a?/?mice. Paraformaldehyde-fixed colon, ileum,
kidney, and liver tissues were dehydrated in ethanol and paraffin embedded. Sections of 6-?m thickness were stained with the anti-CEACAM1a
polyclonal Ab 2456 and counterstained with hematoxylin. (a to d) Tissues from wild-type mice exhibited strong expression at the luminal membrane
surface and crypt colonic and intestinal epithelia (a and b). Proximal tubules of the kidney and bile canaliculi of the liver were also strongly positive
for CEACAM1a in ?/? mice (c and d). (e to h) In contrast, in Ceacam1a?/?mice, colonic (e) or intestinal (f) epithelial cells revealed no staining
of the crypts, in spite of a longer development time. This was also seen for the collecting tubules in the kidney (g) and hepatocytes (h) of
Ceacam1a?/?mice that were not labeled with anti-CEACAM1a Abs.
TABLE 1. Intranasal inoculation of Ceacamla?/?mice on C57BL/6 background with 106PFU of MHV-A59
Viral RNA in brain
Virus titer per g ofe:
2? to 4?
4.7 ? 0.6
5.0 ? 1.3
?100 to 1750 0/5NDd
aThe number of mice represents the total from both 2D2 and 11H11 lines.
bValues are ranges of serum titers (optical density, 0.8) in ELISA of samples from replicate animals; a value of ?100 indicates that the titer was below the range
cGrading system for liver pathology: 0, no lesions: 1, 1 to 10% tissue destruction; 2, 11 to 20% tissue destruction; 3, 21 to 40% tissue destruction; 4, 41 to 100% tissue
dND, not determined.
eValues are geometric mean titers per log10PFU per gram of tissue; a value of ?2 indicates that the titer was below the limit of detection.
10160 HEMMILA ET AL. J. VIROL.
mice demonstrated lesions in their liver at any of the time
points examined (Table 1 and Fig. 3B and C, row I).
Spinal cords from these mice were also prepared for histol-
ogy and analyzed for evidence of demyelination, as indicated
by loss or destruction of myelin detected by Luxol fast blue
staining and the presence of asymetrical lesions (Fig. 3). At 14
and 30 dpi, two of three and four of four wild-type ?/? mice,
respectively, no longer had intact spinal cords and their myelin
sheets were very thin, whereas none of the Ceacam1a?/?mice
showed any abnormalities in these structures at any time point.
Changes in spinal cord histology in the CEACAM1a-positive
?/? mice were generally correlated with the expression of viral
RNA detected in the brains of affected animals. No infectious
virus was detected in the brain or liver of the inoculated ?/?
mice, and no viral RNA from either tissue was detected by
RT-PCR, even when nested PCR primer sets were used.
When Ceacam1a?/?mice were inoculated i.n. with 108PFU
of the same virus (Table 2), none of the ?/? mice showed
clinical signs of disease or liver pathology (Fig. 3C, row I). In
contrast, livers of ?/? mice were completely devastated (liver
pathology grade, 4?) upon i.n. inoculation with just 106viral
PFU. Titers of virus recovered from livers and brains of af-
fected ?/? animals ranged from 8.4 ? 102to 4.6 ? 106PFU/g
of tissue, but no infectious virus was detected in the tissues of
?/? mice. These results indicate that the Ceacam1a?/?mice
were completely resistant to i.n. MHV-A59 infection with at
FIG. 3. (A) Liver and spinal cord lesions in BALB/c ?/? and Ceacam1a partial knockout (p/p) mice on the BALB/c background following i.c.
inoculation with 103PFU MHV-A59. Arrows indicate areas of extensive liver destruction in wild-type ?/? mice (harvested 4 dpi) and
demyelination in spinal cords (harvested 30 dpi) as evidenced by hematoxylin and eosin staining and Luxol fast blue staining. Liver damage was
present in p/p mice but was much less severe than that in the wild-type ?/? mice. Demyelination was also present in both mice but was less severe
in the p/p mice compared to the wild-type ?/? mice. (B) Extensive liver and spinal cord lesions in Ceacam1a?/?mice following i.c. inoculation
with 106PFU MHV-A59. Livers from Ceacam1a?/?and wild-type ?/? C57BL/6 mice were collected 4 dpi, and spinal cords were collected at 30
dpi. Arrows indicate demyelination that was observed in the spinal cords of wild-type ?/? mice. The Ceacam1a?/?mice did not show any signs
of liver damage or spinal cord demyelination. (C) Three-week-old Ceacam1a?/?and wild-type ?/? C57BL/6 mice were i.n. inoculated with 106
PFU of MHV-A59 and sacrificed at 4 dpi. Liver sections were processed for histology, and liver lesions were quantified. Liver lesions were extensive
in wild-type (?/?) mice and absent in the CEACAM1a-deficient mice (?/?). Demyelination was observed in the spinal cords of the wild-type ?/?
C57BL/6 mice but absent in the Ceacam1a?/?mice.
TABLE 2. Intranasal inoculation of Ceacamla?/?mice on C57BL/6
background with MHV-A59a
Virus titer per g ofc
5.5 ? 1.0
5.7 ? 0.9
3.6 ? 0.5
aMice (?/?) were inoculated with 106PFU of virus, and ?/? mice were
infected with 108PFU of virus. Mice from the 2D2 line were tested.
bGrading system for liver pathology: 0, no lesions: 1, 1 to 10% tissue destruc-
tion; 2, 11 to 20% tissue destruction; 3, 21 to 40% tissue destruction; 4, 41 to
100% tissue destruction.
cValues are geometric mean titers per log10PFU per gram of tissue; a value
of ?2 indicates that the titer was below the limit of detection.
VOL. 78, 2004MHV RESISTANCE OF Ceacam1a?/?MICE10161
least 100-fold more than the lethal dose sustained by wild-type
Results of i.c. inoculation of Ceacam1a p/p mice with MHV-
A59. We had previously generated another genetically altered
mouse MHVR mutant (designated p/p mice) in the BALB/c
background. These animals exhibited greatly reduced expres-
sion (?95%) of the CEACAM1a receptor bearing four Ig
domains while the other isoforms containing two Ig domains
were overexpressed (5). When inoculated i.n. with 106PFU of
the MHV-A59 virus, the p/p mice developed small liver lesions
at 3 dpi, compared to their wild-type ?/? littermates that
formed abundant, large lesions and soon succumbed to the
disease. The lesions in the p/p mice completely disappeared at
7 dpi, and these animals completely recovered from the infec-
To gauge the susceptibility of the brains and spinal cords of
p/p mice to MHV-A59 without requiring prior amplification of
virus inoculated at peripheral sites, we directly inoculated 103
PFU of the MHV-A59 virus i.c. and monitored the mice for the
appearance of disease, altered liver and spinal cord histology,
seroconversion, and production of infectious virus or viral
RNA (Table 3). The p/p mice showed only transient and mild
signs of disease, unlike wild-type ?/? mice, which showed
more-extensive neurological signs. Furthermore, 3 of 10 ?/?
mice died between 4 and 14 dpi. Wild-type ?/? mice that had
been inoculated i.c. and sacrificed at 4 dpi showed significantly
altered liver histology (liver pathology grade, 4?) with a larger
number of large lesions than the wild-type mice inoculated i.n.
with the same virus. The Ceacam1a p/p mice examined at 4 dpi
also exhibited liver lesions, but these were consistently fewer
and smaller (2?) than those seen in the wild-type ?/? mice
(Fig. 3A row I). No demyelination of the spinal cord was
observed in any mouse sacrificed at 4 dpi, but viral RNA was
detected in liver and brain of all the ?/? and p/p mice. By 14
dpi, all surviving wild-type ?/? mice had medium-sized liver
lesions (2?), whereas no liver lesions were found in the p/p
mice. On day 14, spinal cord demyelination was observed in all
wild-type ?/? mice and in three of four p/p mice, although the
lesions were less extensive in p/p mice. In wild-type ?/? mice
at 14 dpi, viral RNA was detected in the brains of five of five
mice and the livers of three of five mice, whereas the p/p mice
showed no viral RNA in liver (Table 3). At 30 and 45 dpi, none
of the wild-type ?/? or p/p mice exhibited liver lesions, but
most (four of five ?/? and nine of nine p/p) had spinal cord
demyelination (Fig. 3A, row II). Anti-virus Abs were detected
in the sera of three of three of the p/p mice sacrificed at 30 dpi
and in two of two of the p/p mice sacrificed at 45 dpi. Viral
RNA was detected in brains of two of three wild-type mice at
day 30 and in one of one mouse sacrificed at day 45, but viral
RNA was not detected in brains of p/p mice at these times. No
viral RNA was detected in livers of wild-type ?/? or p/p mice
at days 30 and 45 (Table 3). These experiments show that
MHV-A59 could infect cells in the spinal cords of p/p mice,
although infection caused less-severe demyelination than in
Results of i.c. inoculation of Ceacam1a?/?mice with MHV-
A59. To determine whether the virus infected the p/p mouse
spinal cord via the CEACAM1a receptor, we inoculated
Ceacam1a?/?mice and wild-type ?/? C57BL/6 mice with 106
PFU of MHV-A59 i.c. The results of two independent exper-
iments are summarized in Table 4. Seven of 16 CEACAM1a-
positive ?/? C57BL/6 mice inoculated with virus were sacri-
ficed or died by 4 dpi and had severe liver lesions (4?). At 7
dpi, eight of the nine surviving wild-type ?/? mice were sac-
rificed because they showed severe signs of illness, including
ataxia or paralysis. Livers showed extensive virus-induced dam-
age (4?), and spinal cords of seven of eight mice showed
demyelination. Virus was recovered from the livers and brains
of the wild-type mice, with titers ranging from 102to 105PFU
per gram of liver and 102to 105PFU per gram of brain. On 14
TABLE 3. Intracerebral inoculation of Ceacamla p/p mice on BALB/c background with 103PFU of MHV-A59
Viral RNA in:Virus titer per g ofe:
2? to 4?
1? to 3?
5.3 ? 0.9
3.5 ? 0.4
4.7 ? 0.5
4.1 ? 0.8
1? to 2?
0 to 1?
aThree ?/? mice died between days 4 and 14.
bGrading system for liver pathology: 0, no lesions: 1, 1 to 10% tissue destruction; 2, 11 to 20% tissue destruction; 3, 21 to 40% tissue destruction; 4, 41 to 100% tissue
cValues are ranges of serum titers (optical density, 0.8) in ELISA of samples from replicate animals; a value of ?100 indicates that the titer was below the range
dThe demyelination of the spinal cords of p/p mice was less severe than that of ?/? mice at the same time points.
eValues are geometric mean titers per log10PFU per gram of tissue; a value of ?2 indicates that the titer was below the limit of detection.
10162HEMMILA ET AL.J. VIROL.
dpi, the one surviving wild-type ?/? mouse was sacrificed
because it was moribund. Liver damage (2?) was less extensive
than in animals at 7 dpi, but the spinal cord showed extensive
In marked contrast to the lethal liver and neurological dis-
ease in wild-type ?/? mice, 36 of 36 Ceacam1a?/?mice inoc-
ulated i.c. with MHV-A59 survived (Table 4). All seven ?/?
mice harvested at 4 dpi showed no signs of disease, had no liver
or spinal cord lesions, and had neither viral RNA nor infec-
tious virus in liver or brain. Similar results were obtained with
?/? mice sacrificed at days 14, 30, and 45. Low titers of
anti-virus Abs were detected in a few i.c.-inoculated ?/? mice
at days 7 and 45 (Table 4).
These data show that 3-week-old Ceacam1a?/?mice in the
C57BL/6 background are fully resistant to very high doses of
the MHV-A59 virus administered i.c. or i.n. and indicate that
expression of murine CEACAM1a is essential for MHV-A59
infection of liver or brain of C57BL/6 mice.
The newly generated Ceacam1a?/?mouse lines showed
complete abrogation of expression of the CEACAM1a protein
in all tissues that normally express it. We examined the re-
sponse of two genetically altered mouse models to MHV in-
fection via two different routes of inoculation. We had previ-
ously characterized the p/p mouse response to i.n. inoculation
with MHV-A59 and found that the mice replicated virus in the
liver in small amounts; they showed only mild clinical signs of
disease, recovered, and completely cleared the virus (5). Viral
transmission by the i.n. route first requires viral replication in
the nasal epithelial cells and then dissemination to other or-
gans via either the bloodstream or infected leukocytes or neu-
ral routes (1, 22). In the present study, we showed that i.c.
inoculation also produced hepatic lesions in p/p mice, probably
through viremia. Intracerebral inoculation efficiently induced
small demyelinating lesions in the p/p spinal cords that were
still apparent in mice sacrificed at 45 dpi.
In contrast to the p/p mice that were partially susceptible to
MHV-A59 infection but recovered, mice devoid of the
CEACAM1a receptors (?/?) did not produce any obvious
clinical signs of disease after i.n. inoculation with up to 108
PFU of MHV-A59 virus. The ?/? mice showed no pathology
in liver or spinal cord tissues, and no infectious virus or viral
RNA could be detected in target tissues. Only 2 of the 19
inoculated ?/? mice responded to the inoculum virus as a
foreign antigen and raised anti-virus Abs. These results there-
fore clearly demonstrate that murine CEACAM1a is the sole
receptor for MHV-A59 in C57/BL6 mice in vivo. The two
mouse models discussed herein were produced on different
genetic backgrounds (p/p mice in a BALB/c background and
?/? mice in a C57BL/6 background). Yet, the data are con-
sistent with results obtained with cell lines by a number of
different groups. Furthermore, both the BALB/c and C57BL/6
wild-type mice carry only the Ceacam1a allele and express the
same types of isoforms in the different tissues examined.
Ne ´dellec et al. identified the product of a second Ceacam-
like gene, Ceacam2, as a weak MHV receptor (28). In fact,
hamster cells (BHK) transfected with cDNA encoding murine
CEACAM2 can be infected with MHV-A59 and form multinu-
cleated syncytia (28). However, the virus-neutralizing activity
of the soluble CEACAM2 protein was 10,000-fold lower than
that of the soluble four-Ig-domain-expressing CEACAM1a
TABLE 4. Intracerebral inoculation of Ceacamla?/?mice on C57BL/6 background with 106PFU of MHV-A59
Virus titer per g off
?100 to 125
4.5 ? 0.4
5.2 ? 0.9
?100 to 250 12
aMice tested were from the 2D2 and 11H11 lines; all mice received the same inoculum.
bSeven ?/? mice were sacrificed or died on or before 4 dpi.
cMock-inoculated mice. No MHV-inoculated wild-type mice survived past day 14.
dGrading system for liver pathology: 0, no lesions: 1, 1 to 10% tissue destruction; 2, 11 to 20% tissue destruction; 3, 21 to 40% tissue destruction; 4, 41 to 100% tissue
eValues are ranges of serum titers (optical density, 0.8) in ELISA of samples from replicate animals; a value of ?100 indicates that the titer was below the range
fGeometric mean titer per log10PFU per gram of tissue; a value of ?2 indicates that the titer was below the limit of detection.
VOL. 78, 2004MHV RESISTANCE OF Ceacam1a?/?MICE10163
protein (46). Therefore, the question remained open as to
whether CEACAM2 could serve as an alternate receptor in
vivo and even completely substitute for the CEACAM1a MHV
receptor in Ceacam1a?/?mice. The fact that the Ceacam1a?/?
mice are completely resistant to infection from very high doses
of MHV-A59 strongly suggests that CEACAM2 does not act as
an alternative receptor for MHV in vivo. CEACAM2 is ex-
pressed in wild-type C57BL/6 and BALB/c mice as well as in
Ceacam1a?/?mice (data not shown). However, its tissue dis-
tribution is strikingly different than that of CEACAM1a, being
mostly present in kidney, pancreas, and macrophages and es-
sentially absent in liver and brain (28, 34).
CEACAM1a is a multifunctional protein; abrogating the
expression of this protein leads to a number of phenotypic
abnormalities. Preliminary results obtained from studies of
Ceacam1a?/?mice indicate that they exhibit defects in insulin
clearance from the liver and are hyperinsulinemic and insulin
resistant (Dai et al., unpublished). In addition, the?/? mice
accumulate lipid in their liver, suggesting a serious dysfunction
in the proper utilization or storage of fatty acids and triglyc-
erides and/or possibly a deregulation in their peroxidation (N.
Leung et al., unpublished data). The ?/? mice also show signs
of immune dysfunction. The development of the T lympho-
cytes is normal, the numbers of CD4?versus CD8?T lympho-
cytes are very similar in the thymus and spleen of the ?/? and
?/? mice, and the numbers of IgM-positive B lymphocytes in
the spleen are almost identical in the wild-type ?/? and
knockout ?/? mice. However, the T lymphocytes are func-
tionally impaired in proliferation, with altered cytokine secre-
tion levels (Atallah et al., unpublished). The hepatic or im-
mune dysfunctions might be expected to potentiate viral
infection. In spite of these deficiencies, the C57BL/6
Ceacam1a?/?mice remained entirely resistant to massive
doses of MHV-A59 virus. Thus, murine CEACAM1a proteins
are required for infection of both brain and liver of 3-week-old
C57BL/6 mice with MHV-A59, and the CEACAM1a proteins
are the only receptors for MHV-A59 in these animals.
We thank Michel L. Tremblay for fruitful discussions and the
McGill Transgenic Core Facility personnel for their help. We also wish
to convey our appreciation to the personnel of both the Animal Care
Center of McGill University and the Center for Laboratory Animal
Care at the University of Colorado Health Sciences Center for their
excellent care of the animals.
This work was supported by the Canadian Institutes of Health Re-
search (grant MOP 42501 to N.B.) and the National Institutes of
Health (grant AI25231 to K.V.H.).
Nicole Beauchemin is a senior scientist from the Fonds de la Re-
cherche en Sante ´ du Que ´bec.
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