Microbiol Immunol 2013; 57: 624–632
doi: 10.1111/1348-0421.12078

O R I GIN A L A R T I C L E

Amino acid sequence variations of signaling lymphocyte
activation molecule and mortality caused by morbillivirus
infection in cetaceans
Yui Shimizu1, Kazue Ohishi1, Rintaro Suzuki2, Yuko Tajima3, Tadasu Yamada3, Yuka Kakizoe4,
Takeharu Bando5, Yoshihiro Fujise5, Hajime Taru6, Tsukasa Murayama7 and Tadashi Maruyama1
1

Japan Agency for Marine–Earth Science and Technology (JAMSTEC), 2‐15 Natsushima‐cho, Yokosuka 237‐0061, 2Protein Research Unit,
National Institute of Agrobiological Science, Tsukuba 305‐8602, 3National Museum of Nature and Science, Tsukuba 305‐0005, 4Port ofNagoya Public Aquarium, Nagoya 455‐0033, 5Institute of Cetacean Research, Tokyo 104‐0055, 6Kanagawa Prefectural Museum of
Natural History, Odawara 250‐0031 and 7Tokai University, Shizuoka 424‐0092, Japan

ABSTRACT
Morbillivirus infection is a severe threat to marine mammals. Mass die‐offs caused by this infection haverepeatedly occurred in bottlenose dolphins (Turiops truncatus) and striped dolphins (Stenella
coeruleoalba), both of which belong to the family Delphinidae, but not in other cetaceans. However,
it is unknown whether sensitivity to the virus varies among cetacean species. The signaling lymphocyte
activation molecule (SLAM) is a receptor on host cells that allows morbillivirus invasion andpropagation. Its immunoguloblin variable domain‐like (V) region provides an interface for the virus
hemagglutinin (H) protein. In this study, variations in the amino acid residues of the V region of
26 cetacean species, covering almost all cetacean genera, were examined. Three‐dimensional (3D) modelsof them were generated in a homology model using the crystal structure of the marmoset SLAM andmeasles virus H protein complex as a template. The 3D models showed 32 amino acid residues on theinterface that possibly bind the morbillivirus. Among the cetacean species studied, variations were foundat six of the residues. Bottlenose and striped dolphins have substitutions at ﬁve positions (E68G, I74V,
R90H, V126I, and Q130H) compared with those of baleen whales. Three residues (at positions 68, 90 and130) were found to alternate electric charges, possibly causing changes in afﬁnity for the virus. This study
shows a new approach based on receptor structure for assessing potential vulnerability to viral infection.
This method may be useful for assessing the risk of morbillivirus infection in wildlife.
Key words cetacean, morbillivirus, receptor, signaling lymphocyte activation molecule.

Marine mammals face many risks from recent climatechange, environmental contaminants and infectious
diseases. Morbillivirus infection is one of the most severe
threats to marine mammals such as cetaceans and seals.
The morbillivirus is a member of the family Paramyxoviridae. Four species, namely measles virus (MV) in
humans, rinderpest virus (RPV) in cows, peste des petits
ruminants virus in sheep and goats, and canine distemper

virus in dogs, have been identiﬁed in terrestrial
mammals (1). Since the late 1980 s, morbilliviruses haverepeatedly caused mass die‐offs of marine mammals (2, 3).
Two novel morbillivirus species, phocine distemper virus
and cetacean morbillivirus (CMV), have been isolated
from affected dead seals and cetaceans, respectively (4–6).
The ﬁrst isolation of CMV was from several harbor
porpoises (Phocoena phocoena) with pathological evidence

Correspondence
Kazue Ohishi, Japan Agency for Marine‐Earth Science and Technology (JAMSTEC), 2‐15 Natsushima‐cho, Yokosuka 237‐0061, Japan.
Tel: þ81 46 867 9524; fax: þ81 46 867 9525; email: oishik@jamstec.go.jp
Received 25 March 2013; revised 30 May 2013; accepted 25 June 2013.
List of Abbreviations: 3D, three‐dimensional; C, constant‐2 domain‐like; CMV, cetacean morbillivirus; H, hemagglutinin; MV, measles virus; RPV,
rinerpest virus; SINE, short interspersed element; SLAM, signaling lymphocyte activation molecule; V, variable domain‐like.

624© 2013 The Societies and Wiley Publishing Asia Pty Ltd

Variations in SLAM in cetacean speciesof the virus stranded on the Irish coast in 1988 (5). CMV
induced a severe mass die‐off of the striped dolphin(Stenella coeruleoalba) population on the Mediterranean
coast of Spain, which rapidly spread throughout thewestern Mediterranean sea, including the coasts ofFrance, Italy, Greece and Turkey (6). Retrospective
studies showed that CMV killed several thousand
bottlenose dolphins (Tursiops truncatus) along the Atlantic
coast of the USA in 1987–1988, and several hundred
dolphins in the Gulf of Mexico during 1993–1994 (7, 8).
Thus, in the past few decades, CMV has caused mass die‐offs in cetaceans, particularly in dolphins belonging to theDelphinidae family. However, it is still unknown whether
sensitivity to the morbillivirus differs among various
cetacean species.
All morbilliviruses are lymphotropic and lymphoidtissues are the major sites of viral replication. SLAM
(CD150) is the major cellular receptor for the entry andpropagation of morbilliviruses in humans, cows anddogs (9, 10). It is expressed on various immune cells
such as activated T cells, B cells, mature dendritic cells
and thymocytes (11). The distribution of SLAM explains
the viral lymphotropism and immunosuppressive
nature of the morbillivirus and strongly suggests that
SLAM is the universal receptor for entry of morbilliviruses into mammalian host cells. Because previous
studies of CMV‐infected cetaceans have shown thatthese viruses are also mainly distributed in lymphoidtissues, and because the pathology in these animals
is reportedly very similar to that of other morbillivirus‐
infected animals, cetacean SLAM on immune cells arethought to function as receptors for CMV (12, 13).
SLAM is the principal member of the SLAM family,
which belongs to the immunoglobulin superfamily,
and shares a membrane‐distal immunoglobulin V
region and a membrane‐proximal immunoglobulin C
region in its extracellular space (14). It acts as a self‐
ligand and forms a homophilic dimer by weak binding
via the V domain (15). The V domain also has aninterface for binding morbilliviruses (16). The viral H
protein has a strong afﬁnity for the V domain of
SLAM, which is 400‐fold greater than that for self‐
ligand interaction (17). Binding between SLAM and the
H protein triggers subsequent cell fusion events via
viral fusion protein, allowing viral invasion of hostcells (18–20). Substitution experiments on the aminoacid residues of the human SLAM interface haveshown that some residue substitutions lead to a loss ora reduction of, or sometimes an increase in, viral
infectivity, indicating that these residues are key forboth binding afﬁnity and viral infectivity (16, 18, 19).
Taken together, these ﬁndings raise the possibility
that, among cetaceans, there are variations in the amino© 2013 The Societies and Wiley Publishing Asia Pty Ltd

acid residues on the interfaces of SLAMs and that some ofthem may be key residues for binding afﬁnity andsensitivity to the virus.
We previously determined the complete nucleotide
sequences of SLAMs from three different marine
mammal groups, namely cetaceans, pinnipeds andsirenians, and generated 3D homology models using
the human NK, T, and B cell antigen (NTB‐A) molecule, a
SLAM family protein, as a template (21, 22). We
suggested that some amino acid residues on the interface
region of these marine mammal SLAMs are key residues
for both viral binding and host‐virus speciﬁcity (21). The
crystal structure of the complex of MV‐H protein andthe V region of marmoset (Saguinus oedipus) SLAM
(marSLAM) was recently determined (18). Crystal
structural analysis showed that this complex contains
four important binding sites for the virus (sites 1–4), andthat an electric interaction plays an important role in thebinding.
In the present study, we examined variations in aminoacid residues in the interface region of SLAM in various
species of cetaceans, including six families of toothed
and three families of baleen whales. Their 3D models
were generated by homology modeling using the
crystal structure of the marSLAM‐MV‐H complex and
NTB‐A as templates. In comparing the key residues
contributing to viral binding, we discuss the sensitivityof cetaceans to CMV infection and a possible meansof assessing the risk of morbillivirus infection incetaceans.

MATERIALS AND MEHODS
Cetacean tissue samples
Tissue samples were collected from 19 species of toothed
whales and 7 species of baleen whales (Table 1). Species
identiﬁcation was based on traditional morphologic
classiﬁcations (23). Beluga whale (Delphinapterus leucas)
tissue was taken from a dead captive animal (Port ofNagoya Public Aquarium). Tissues from common minke(Balaenoptera acutorostrata), sperm (Physeter macrocephalus),
Bryde's (Balaenoptera brydei) and Sei whales (Balaenoptera
borealis) were collected legally in 2000 or 2001 during theWestern North Paciﬁc Phase II (JARPN II) of the Japanese
Whale Research Program, which was authorized by thegovernment of Japan. JARPN II is in full compliance withthe relevant international treaty, namely the International
Convention for the Regulation of Whaling. Other tissues
were taken from dead stranded whales along the coast ofJapan. They were collected as early as possible during
postmortem investigations conducted by the National
Museum of Nature and Science and stored at 20 or
80°C until use.
625

Y. Shimizu et al.

Table 1. List of cetacean samples
Species
Odontoceti
Delphinidae
Bottlenose dolphin
Indo‐Pacific bottlenose dolphin
Striped dolphin
Melon‐headed whale
Pygmy killer whale
False killer whale
Risso's dolphin
Short‐finned pilot whale
Phocoenidae
Harbor porpoise
Finless porpoise
Dall's porpoise
Monodontidae
BelugaZiphiidae
Stejneger's beaked whale
Blainville's beaked whale
Ginkgo‐toothed beaked whale
Hubb's beaked whale
Kogiidae
Pygmy sperm whale
Dwarf sperm whale
Physeteridae
Sperm whale
Mysticeti
Balaenopteridae
Fin whale
Common minke whale
Bryde's whale
Sei whale
Humpback whale
Balaenidae
North Pacific right whale
Eschrichtiidae
Gray whale

Scientific name

Accession no.

Tursiops truncatus
Tursiops aduncus
Stenella coeruleoalba
Peponoceplala electra
Feresa attenuata
Pseudorca crassidens
Grampus griesus
Globicephala macrorhynchus

AB612203
AB612204
AB612205
AB612206
AB612207
AB612208
AB612209
AB612210

Phocoena phocoena
Neophocaena phocaenoides
Phocoenoides dalli

AB612211
AB612212
AB612213

Delphinapterus leucas

AB612214

Mesoplodon stejnegeri
Mesoplodon densirostris
Mesoplodon ginkgodens
Mesoplodon carlhubbsi

AB612215
AB612216
AB612217
AB612218

Kogia breviceps
Kogia sima

AB612219
AB612220

Physeter macrocephalus

AB612221

Balaenoptera physalus
Balaenoptera acutorostrata
Balaenoptera brydei
Balaenoptera borealis
Megaptera novaeangliae

AB612222
AB612223
AB612224
AB612225
AB612226

Eubalaena japonica

AB612227

Eschrichtius robustus

AB612228

Determination of nucleotide sequences of
the interface on cetacean signaling
lymphocyte activation molecules
Deoxyribonucleic acid was extracted from whale tissues
using a commercial DNA extraction kit (DNeasy Tissue
Kit; Qiagen, Boston, MA, USA). For detection of the
SLAM exon 2 coding the V region, the forward primer(dol‐SLAM‐DF3: 50 ‐GTGAGGGCTCAACGAATTGC‐
30 ) was designed based on the nucleotide sequences of
SLAM in killer whales (Orcinus orca; AB428367) andPaciﬁc white‐sided dolphins (Lagenorhynchus obliquidens;
AB428366). The reverse primer was dol‐SLAM‐DR2(50 ‐CATAGAGCTTCAGCTGCAGG‐30 ) (21). Using thisprimer set, PCR was performed. Ampliﬁcation was
carried out in a thermal cycler under the following
conditions: 96°C for 1 min; 30 cycles of 96°C/20 s, 57°C/
626

30 s, and 72°C/1 min; followed by a ﬁnal 10 min 72°C
extension. The nucleotide sequences of the DNA
fragments produced were determined using the dyeterminator method.
Homology modeling of the three‐dimensional structure of signaling
lymphocyte activation molecule
The Protein Data Bank entries for marSLAM in thecomplex with the measles virus H protein (3ALW: A–D
chains, 3ALX: A–D chains, 3ALZ: B chain (18)) and forhuman NTB‐A (21F7: A–D chains (22)) were used as the
template structures. Three‐dimensional models wereconstructed using the MODELLER 9.10 program (24)and visualized using PyMOL 1.5.0.3 (Schrödinger LLC)
and PovRay (Persistence of Vision).
© 2013 The Societies and Wiley Publishing Asia Pty Ltd

Variations in SLAM in cetacean species

RESULTS
Variations in the deduced amino acidresidues of cetacean signaling lymphocyte
activation molecules
Polymerase chain reaction ampliﬁcations of DNA
samples from all 26 cetacean species studied produced
DNA fragments approximately 300 bp in length. The
sequence data have been submitted to the GeneBank
database; the accession numbers assigned are listed inTable 1. Among the cetaceans, variations were found at
the nucleotide level at 30 nucleotide positions in theampliﬁed and sequenced DNA fragments. Replacements of the deduced amino acid residues were detected
at 13 residue positions (amino acid residue positions 32,
50, 51, 53, 66, 68, 71, 74, 82, 90, 103, 126 and 130;
Fig. 1).
Three‐dimensional models of cetaceansignaling lymphocyte activation moleculeinterfaces with morbillivirus
A 3D model of the killer whale SLAM with its completeamino acid sequence was generated. This model showedthat it contains a pair of globular immunoglobulin‐like V
and C regions, and that the V region consisted of twob‐sheets, as in previous models (21) (Fig. 2a). The front
sheet in the present models contains four anti‐parallelb‐strands and appears to provide an interface for themorbillivirus (Fig. 2b). Comparison with the interface of
marmoset SLAM, shown in Figure 2c, indicates that the
3D structures of the interfaces of killer whale andmarmoset are similar. The 12 residues involved in thefour binding sites in the marSLAM‐MV‐H complex (Site
1: 77, 90, Site 2: 61, 63, and 123, Site 3: 127‐131, and Site
4: 75 and 119) are depicted in different colors with blackside chains in the representation of the marmoset SLAM
interface (Fig. 2c). In addition to the twelve residues on
the cetacean interface that correspond with those ofmarmoset SLAM, the residues with protruding side
chains are likely components of virus binding. Thus, the32 amino acid residues on the cetacean interface of the 3Dmodel are thought to contribute to morbillivirus binding(Fig. 2b). When the 32 residues were compared amongcetacean groups, variations were found at the six residue
positions 68, 74, 82, 90, 126 and 130 (Table 2, bold andunderlined). The substitutions at two positions, 74 and126, are between similar characteristic residues, that is,Fig. 1. Deduced amino acid sequences of the SLAM‐V region in 26 cetacean species. The position number of the residues is that of the complete
SLAM protein of the killer whale. The sequence data of the killer whale was obtained from Reference 21 (Accession no. AB428367) and the othersequences were determined in the present study. The residues corresponding to the PCR primers are not shown.

© 2013 The Societies and Wiley Publishing Asia Pty Ltd

627

Y. Shimizu et al.

Fig. 2. Ribbon diagram of the 3D model of the killer whale SLAM extracellular region. In (a), the blue and green models show the two SLAM
extracellular domains that form a homophilic dimer. The b‐strands are indicated by blue and green arrows and the disulﬁde bonds are shown as
yellow bars. The thick red arrow indicates the direction of view of the interface for morbillivirus binding as shown in (b). In (b), the position
numbers of the amino acid residues that possibly interact with the viral H protein are shown. The side chains are shown with the atoms colored(black for carbons, blue for nitrogens and red for oxygens). For a reference, the marmoset SLAM interface is shown in (c). The position numbers of
the residues involved in the four binding sites of marSLAM‐MV‐H complex are differently colored as follows: Site 1, mint green; Site 2, cyan; Site3, red; Site 4, yellow and all with black side chains.

hydrophobic and uncharged residues, whereas the otherfour substitutions at of the interface were accompanied bycharge alterations. The SLAM interfaces of toothed andbaleen whales are depicted in Figure 3 and show that thesix residue positions are located at the edge of theinterfaces (Fig. 3).
Cetacean signaling lymphocyte activation
molecule substitutions traced in thephylogeny
The six substitutions of the residues on the cetacean
phylogenetic tree were plotted based on the SINE method,which is widely accepted in the classiﬁcation of thecetaceans (Fig. 4) (25–27). The cow (Bos taurus, accession
no. AF329970), a member of the order Cetartiodactyla inwhich the cetaceans are nested (25), was used as theoutgroup. The remaining 26 of the 32 residues on theinterface were found to be completely identical incetaceans and cows. The 32 residues were nearly identicalamong baleen whale SLAMs, although substitutions were
frequently found in the residues of toothed whales,
particularly in Delphinidae (Fig. 4). Compared with theresidues in baleen whales such as the gray whale, ﬁve
substitutions (E68G, I74V, R90H, V126I and Q130H)
were found in bottlenose and striped dolphins; mass die‐offs have been recorded in both of these species. Three ofthese substitutions (E68G, R90H and Q130H) result incharge changes of the interface (Fig. 4).
628

DISCUSSION
In this study, we compared the SLAM‐V domains ofvarious cetacean species; species not studied included
river dolphins such as the Indian river dolphin(Platanista gangetica) and La Plata dolphin (Pontoporia
blainvillei) and the pygmy right whale (Caperea marginata; Table 1). These domains potentially function
as binding interfaces for morbilliviruses. Three‐dimensional homology models of cetacean SLAMs
showed that 32 amino acid residues possibly bind to thevirus on the interface and that, among the cetacean
species studied, variations occur at six amino acidresidue positions (Table 2). These six positions arelocated at the edge of the interface, suggesting that theresidues in the central region play an essential role in a
primary immunological function of SLAM and cannotchange (Fig. 3).
Because the nucleotide or amino acid sequences of the
SLAM‐V region are too short to reconstruct a reliablephylogenetic tree, we plotted the substitutions on aphylogenetic tree constructed using the SINE method(Fig. 4), which classiﬁes cetaceans in a manner consistent
with their morphological features (25–27). Comparedwith baleen whales, ﬁve residue substitutions were foundin bottlenose and striped dolphins, the species in whichmost cetacean mass die‐offs have reportedly occurred(6–8). Three of the ﬁve residues (G68, H90 and H130)
introduced an alteration in the charge. SLAM genes are© 2013 The Societies and Wiley Publishing Asia Pty Ltd

Variations in SLAM in cetacean species

Table 2. Amino acid residues on cetacean SLAM interfaces that are possibly involved in regulating the binding and speciﬁcities of morbillivirus
Baleen whales†

Toothed whales

58
60
61‡
63‡
65
67
68
69
72
73
74
75‡
76
77‡
80
82
84
85
87
90‡
92
117
119‡
121
123‡
125
126
127‡
128‡
129‡
130‡
131‡

Delphinidae

Phocoenidae

Monodontidae

Ziphiidae

KoggidaePhysteridae

Balaenopteridae†

Balanidae†

Eschrichtiidae†

K
I
H
L
T
A
G
S
D
T
V
K
K
K
S
D or Y
R
K
D
H
L
W
F
S
E
N
I
S
V
Q
Q or H
F

K
I
H
L
T
A
E
S
D
T
T
K
K
K
S
D
R
K
D
R
L
W
F
S
E
N
I
S
V
Q
Q or H
F

K
I
H
L
T
A
E
S
D
T
I
K
K
K
S
D
R
K
D
R
L
W
F
S
E
N
I
S
V
Q
Q
F

K
I
H
L
T
A
E
S
D
T
I
K
K
K
S
D
R
K
D
H
L
W
F
S
E
N
V
S
V
Q
Q
F

K
I
H
L
T
A
E
S
D
T
I or T
K
K
K
S
D
R
K
D
R
L
W
F
S
E
N
I
S
V
Q
Q
F

K
I
H
L
T
A
E
S
D
T
I
K
K
K
S
D
R
K
D
R
L
W
F
S
E
N
V
S
V
Q
Q
F

K
I
H
L
T
A
E
S
D
T
I or V
K
K
K
S
D
R
K
D
R
L
W
F
S
E
N
I or V
S
V
Q
Q
F

K
I
H
L
T
A
E
S
D
T
I
K
K
K
S
D
R
K
D
R
L
W
F
S
E
N
V
S
V
Q
Q
F

K
I
H
L
T
A
E
S
D
T
I
K
K
K
S
D
R
K
D
R
L
W
F
S
E
N
V
S
V
Q
Q
F

Bold and underlined: residue positions that vary among cetacean species.
†, Baleen whales. ‡, Residues on cetacean SLAMs that correspond to those in the binding sites in the complex of marSLAM V and MV‐H.

comparatively well conserved among mammals and the
32 amino acid residues on the interface have highhomology between evolutionarily closely related animals.
For example, the SLAMs in two pinniped species, thespotted seal (Phoca largha, AB428368) and walrus(Odobenus rosmarus, AB428369), are completely identicalin those residues. The SLAMs in seals and canines (Canis
lupus familiaris, AF325357) only differ in two residues, andthose two residues are substituted with similar characteristic residues (21, 28). This similarity may explain why
mass die‐offs of Baikal and Caspian seals have beencaused by canine distemper virus infection (29–31).
Considering these ﬁndings, the substitutions in the twodolphins belonging to the Delphinidae family appear tooccur frequently. This suggests that the afﬁnity of suchdolphin SLAMs for the morbillivirus is higher than that© 2013 The Societies and Wiley Publishing Asia Pty Ltdof other cetacean SLAMs (Table 2, Fig. 3). That afﬁnity
may intensify viral infectivity and tissue‐to‐tissue
propagation of the virus in the host animal. McCarthy
et al. reported no amino acid residue variations among
regional populations of the European harbor seal (Phoca
vitulina) in the SLAM exon 2 and exon 3 regions (32),although the mortality rates varied in mass die‐offs
occurring in 1988 and 2002 in these seals (4, 33). Suchhomology of SLAM genes within the same species isunsurprising, because the present study has shown that
SLAM amino acid sequences are relatively highly
conserved at genus or family levels. Other ecological or
epidemiological factors may have affected the variation in
mortality rate of European harbor seal populations.
However, it is interesting that, among seal populations, asingle‐nucleotide polymorphism is present only in exon 2629

Y. Shimizu et al.

Fig. 3. Interfaces of the 3D models of bottlenose dolphin SLAM (a) and gray whale SLAM (b). The position numbers of the amino acid residues
that possibly interact with the viral H protein are shown. The amino acid residues that differ between these two cetacean SLAMs are indicated inblack, whereas shared residues are in blue. Their side chains are shown with the atoms colored (black for carbons, blue for nitrogens and red foroxygens). The amino acid residues corresponding to the PCR primers are indicated by red ribbons.

encoding the V region (32). Accumulation of single‐
nucleotide polymorphisms may promote alteration in theamino acid sequence and in features of the SLAM
interface.
Hashiguchi et al. have recently reported that four bindingsites (sites 1–4) are important in the crystal structure of thecomplex between the marSLAM‐V domain and MV‐H
protein (18). The 12 residues on the marmoset SLAM
interface are reportedly involved in the binding sites (18). Allof the residues on the cetacean SLAM interfaces corresponding to these twelve marmoset SLAM residues were
found in the possible virus‐binding 32 residues (Fig. 2,
Table 2). Among the three residue positions with chargealterations among cetaceans (68, 90 and 130), the residues at
90 and 130 of marSLAM are involved in the four sites. At site
1, the positively charged residue R90 of marSLAM interacts
with D507, a negatively charged residue of MV‐H, which isconserved in all morbilliviruses, including CMV. Residue
R130 of marSLAM is involved in both sites 3 and 4.
Replacement of residue R130 with S130 dramatically
impairs the ability to bind to MV‐H (19). At site 4, R130
binds to another SLAM residue, the negatively charged E75,
via a salt bridge. On the other hand, cetacean SLAMs haveresidues K75, H/R90 and H/Q130 instead of the E75, R90
and R130 of marSLAM. Because these cetacean residues aredifferently charged from those of marSLAM, they may
interact with CMV in a different manner than that between
the MV‐H and marSLAM complex. However, the difference
among cetacean species in these residues may affect the
afﬁnity of the SLAM interface for CMV. To determine theeffect of each substitution in cetacean SLAM on its binding
afﬁnity for CMV, crystal structural analyses of the complex
of cetacean SLAM‐V and CMV‐H, or binding afﬁnity
experiments between the two proteins, are needed. Further,a CMV infection experiment using cetaceans, or alterna630

tively using knockin mice expressing cetacean SLAMs, will
give further insight into the role of cetacean SLAMs in CMV
infectivity among cetaceans (34).
The similarity of the 32 residues on the interface of
cetacean SLAMs to those of cow SLAMs is noteworthy.
When the residues of cow SLAM were compared withthose of human or dog SLAM, 11 and 8 substitutions
were observed, respectively (28). However, those of
baleen whales such as the gray whale have only two
substitutions (V74I and H130Q) that differ from thoseof cows (Fig. 4). This similarity is expected because
cetaceans and artiodactyla such as cows and sheep are
evolutionarily related. Interestingly, the 32 residues ofthe bottlenose dolphin have only three substitutions(E68G, R90H and V126I) that differ from those of thecow, although they have ﬁve substitutions (E68G,
I174V, R90H, V126I and Q130H) that differ from thoseof the gray whale (Fig. 3). This is because two
substitutions (I74 and Q130) that occurred in whale
SLAMs after divergence from cows reverted to the same
residues as those of the cow (V74 and H130) in dolphin
SLAMs (Fig. 4). The strong similarities of the residues
on the interface may indicate that CMV can use cow
SLAM. In addition, RPV may also be able to infectcetaceans. However, it is unlikely that cetaceans would
encounter RPV in nature. This possibility should bestudied in future.
In addition to other cellular factors involved in viral
replication, many ecological factors, including distribution, migration, population density and reproductive
behavior, are probably important in the epidemiology ofmass die‐offs. Environmental pollutants may havesynergistic effects on cetacean mortality. However, among
the numerous possible factors, we focused on the afﬁnity
between the morbillivirus and its receptor to estimate the© 2013 The Societies and Wiley Publishing Asia Pty Ltd

Variations in SLAM in cetacean species

Fig. 4. Residue substitutions at six positions plotted on a phylogenetic tree based on the SINE method. The topology of the phylogenetic tree
was based on previous reports using the SINE method (25–27). Information for the cow (Bos taurus, accession no. AF329970) was used as theoutgroup. The arrowheads and numbers at the branches of the tree indicate the occurrence of substitutions and numbers of amino acid residues:
68, E68G (CGA); 74, V74I (ATC); 74', I74T (ACC); 74“, I74V (GTC); 82, D82Y (TAT); 90, R90H (CAC); 90', R90H (CAT); 126, V126I (ATT); 130,
H130Q (CAA); and 130', Q130H (CAC) (the changed nucleotides are underlined). The two substitutions that reverted (74” and 130') are shown inred. Dolphins in which mass die‐offs have been recorded are indicated with asterisks.

potential vulnerability of cetaceans to viral infection.
Further studies on SLAM structure in various forms ofwildlife may be useful for assessing their risk ofmorbillivirus infection in nature.

ACKNOWLEDGMENT
This study was partially supported by the Japan Society
for the Promotion of Science.

DISCLOSURE
All authors have no conﬂicts of interest to disclose.
© 2013 The Societies and Wiley Publishing Asia Pty Ltd

REFERENCES
1. Grifﬁn D.E. (2001) Measles virus. In: Knipe D.M., Howley, P.M.,
eds. Field's virology, 3rd edn. Philadelphia, Pennyslvania:
Lippincott Williams & Wilkins Press, pp. 1401–41.
2. Barrett T., Rima B.K. (2002) Molecular biology of morbillivirus
diseases of marine mammals. In: Pfeiffer C.J., ed. Molecular andcell biology of marine mammals. Malabar, Florida: Krieger
Publishing, pp. 161–72.
3. Di Guardo G., Marruchella G., Agrimi U., Kennedy S. (2005)
Morbillivirus infections in aquatic mammals: a brief overview.
J Vet Med Series A 52: 88–93.
4. Mahy B.W.J., Barrett T., Evans S., Anderson E.C., Bostock C.J.
(1988) Characterization of a seal morbillivirus. Nature 336:
115–6.

631

Y. Shimizu et al.

5. Kennedy S., Smyth J.A., Cush P.F., McCullough S.J., Allan G.M.,
McQuaid S. (1988) Viral distemper now found in porpoises.
Nature 336: 21.
6. Domingo M., Ferrer L., Pumaorola M., Marco A., Plana J.l.,
Kennedy S., McAliskey M., Rima B.K. (1990) Morbillivirus indolphins. Nature 348: 21.
7. Lipscomb T.P., Schulman F.Y., Moffett D., Kennedy S. (1994)
Morbillivirus disease in Atlantic bottlenose dolphins (Tursiops
truncatus) from the 1987–1988 epizootic. J Wildlife Dis 30: 567–71.
8. Lipscomb T.P., Kennedy S., Moffett D., Krafft A., Klaunberg B.A.,
Lichy J.H., Regan G.T., Worthy G.A., Taubenberger J.K. (1996)
Morbillivirus epizootic in bottlenose dolphins of the Gulf of
Mexico. J Vet Diagn Invest 8: 283–90.
9. Tatsuo H., Ono N., Tanaka K, Yanagi Y. (2000) SLAM (CDw150)
is a cellular receptor for measles virus. Nature 406: 893–7.
10. Tatsuo H., Ono N., Yanagi Y. (2001) Morbilliviruses use signaling
lymphocyte activation molecules (CD150) as cellular receptors.
J Virol 75: 5842–50.
11. Veillette A. (2010) SLAM‐family receptors: Immune regulatorswith or without SAP‐family adaptors. Cold Spring Harbor Perspect
Biol 2: a002469.
12. Kennedy S. (1998) Morbillivirus infections in aquatic mammals.
J Comp Pathol 119: 201–25.
13. Kennedy S., Lipscomb T., Shulman Y. (2002)
Immunohistochemistry of morbillivirus infections in seals,
harbor porpoises, and bottlenose dolphins. In: Pfeiffer C.J., ed.
Molecular and Cell Biology of Marine Mammals. Malabar,
Florida: Krieger Publishing, pp. 178–85.
14. Veillette A., Cruz‐Munoz M., Zhong M. (2006) SLAM family
receptors and SAP‐related adaptors: matters arising. Trends
Immunol 27: 228–34.
15. Mavaddat N., Mason D.W., Aikinson P.D., Evans E.J., Gilbert
R.J., Stuart D.I., Fennelly J.A., Barclay A.N., Davis S.J., Brown
M.H. (2000) Signaling lymphocytic activation molecule(CDw150) is homophilic but self‐associates with very low
afﬁnity. J Biol Chem 275: 28,100–9.
16. Ono N., Tatsuo H., Tanaka K., Minagawa H., Yanagi Y. (2001) V
domain of human SLAM (CDw150) is essential for its function
as a measles virus receptor. J Virol 75: 1594–600.
17. Hashiguchi T., Kajikawa M., Maita N., Takeda M., Kuroki K.,
Sasaki K., Kohda D., Yanagi Y., Maenaka K. (2007) Crystal
structure of measles virus hemagglutinin provides insight into
effective vaccines. Proc Natl Acad Sci USA 104: 19,535–40.
18. Hashiguchi T., Ose T., Kubota M., Maita N., Kamishikiryo J.,
Maenaka K., Yanagi Y. (2011) Structure of the measles virus
hemagglutinin bound to its cellular receptor SLAM. Nature Struct
Mol Biol 18: 135–41.
19. Hashiguchi T., Maenaka K., Yanagi Y. (2011) Measles virus
hemagglutinin: structural insights into cell entry and measles
vaccine. Frontiers Microbiol 2: Article 247.
20. Plemper R.K., Brindley M.A., Iorio R.M. (2011) Structural andmechanistic studies of measles virus illuminate paramyxovirus
entry. PLoS Path 7: e1002058.
21. Ohishi K., Ando A., Suzuki R., Takishita K., Kawato M.,
Katsumata E., Ohtsu D., Okutsu K., Tokutake K., Miyahara H.,

632

Nakamura H., Murayama T., Maruyama T. (2010) Host‐virus
speciﬁcity of morbilliviruses predicted by structural modeling of
the marine mammal SLAM, a receptor. Comp Immunol Microbiol
Infect Dis 33: 227–41.
22. Cao E., Ramagopal U.A., Fedorov A., Fedorov E., Yan Q., Lary
J.W., Cole J.L., Nathenson S.G., Almo S.C. (2006) NTB‐A
receptor crystal structure: insights into homophilic interactionsin the signaling lymphocytic activation molecule receptor family.
Immunity 25: 559–70.
23. Committee on Taxonomy 2012. List of marine mammal species
and subspecies, Society for Marine Mammalogy HP, http://www.
marinemammalscience.org/index.php?
option¼com_content&view¼article&id¼645&Itemid¼340.
24. Šali A., Blundell T.L. (1993) Comparative protein modelling by
satisfaction of spatial restraints. J Mol Biol 234: 779–815.
25. Shimamura M.H., Yasue K., Ohshima H., Abe H., Kato T.,
Kishiro T., Goto M., Munechika I., Okada N. (1997) Molecularevidence from retroposons that whales form a clade within even‐toothed ungulates. Nature 388: 666–70.
26. Nikaido M., Matsuno F., Hamilton H., Brownell R.L., Jr, Cao Y.,
Ding W., Zuoyan Z., Shedlock A.M., Fordyce R.E., Hasegawa M.,
Okada N. (2001) Retroposon analysis of major cetacean lineage:
The monophyly of toothed whales and the paraphyly of riverdolphins. Proc Natl Acad Sci USA 98: 7384–9.
27. Nikaido M., Hamilton H., Makino H., Sasaki T., Takahshi K.,
Goto M., Kanda N., Pastene L.A., Okada N. (2006) Baleen whale
phylogeny and a past extensive radiation event revealed by SINE
insertion analysis. Mol Biol Evol 23: 866–73.
28. Ohishi K., Suzuki R., Maruyama T. (2012) Host‐virus speciﬁcityof the morbillivirus receptor, SLAM, in marine mammals: risk
assessment of infection based on three‐dimensional models. In:
Romero A., Keith E.A., eds. New Approaches to the Study ofMarine Mammals. Rijeka: InTech Press, pp. 183–204.
29. Mamaev L.V., Visser I.K., Belikov S.I., Denikina N.N., Harder T.,
Goatley L., Rima B., Edginton B., Osterhaus A.D., Barrett T.
(1996) Canine distemper virus in Lake Baikal seals (Phoca
sibirica). Vet Rec 138: 437–9.
30. Forsyth M.A., Kennedy S., Wilson S., Eybatov T., Barrett T. (1998)
Canine distemper virus in a Caspian seal. Vet Rec 143: 662–4.
31. Kennedy S., Kuiken T., Jepson P.D., Deaville R., Forsyth M.,
Barrett T, van de Bildt M.W., Osterhaus A.D., Eybatov T., Duck
C., Kydyrmanov A., Mitrofanov I., Wilson S. (2000) Mass die‐offof Caspian seals caused by canine distemper virus. Emerg Infect Dis
6: 637–9.
32. McCarthy A.J., Shaw M.A., Jepson P.D., Brasseur S.M., Reijinders
P.J., Goodman S.J. (2011) Variation in European harbour seal
immune response genes and susceptibility to phocine distemper
virus (PDV). Infect Genet Evol 11: 1616–23.
33. Jensen T., van de Bildt M., Dietz H.H., Andersen T.H., Hammer
A.S., Kuiken T., Osterhaus A. (2002) Another phocine distemper
outbreak in Europe. Science 297: 209.
34. Ohno S., Ono N., Seki F., Takeda M., Kura S., Tsuzuki T., Yanagi
Y. (2007) Measles virus infection of SLAM (CD150) knockinmice reproduces tropism and immunosuppression in humaninfection. J Virol 8: 1650–9.

© 2013 The Societies and Wiley Publishing Asia Pty Ltd