Advance Publication
The Journal of Veterinary Medical Science
Accepted Date: 12 Mar 2013 J-STAGE Advance Published Date: 26 Mar 2013 1 Virology 2 3 NOTE 4 5 Title: 6 Function of feline signaling lymphocyte activation molecule as a receptor of canine 7 distemper virus 8 9 Authors: 10 Yuka HARA1), Junko SUZUKI1), Keita NOGUCHI1), Yutaka TERADA1), Hiroshi 11 SHIMODA1), Takuya MIZUNO2) and Ken MAEDA1), * 12 13 Laboratories of 1)Veterinary Microbiology and 2)Veterinary Internal Medicine, Joint 14 Faculty of Veterinary Medicine, Yamaguchi University, 1677-1 Yoshida, Yamaguchi 15 753-8515, Japan 16 17 18 Running head: 19 FELINE SLAM AS A RECEPTOR OF CDV 20 21 *CORRESPONDENCE TO: MAEDA, K., Laboratory of Veterinary Microbiology, 22 Joint Faculty of Veterinary Medicine, Yamaguchi University, 1677-1 Yoshida, 23 Yamaguchi 753-8515, Japan 24 E-mail: [email protected]
25
1
26 ABSTRACT
27 Morbilliviruses use signaling lymphocyte activation molecule (SLAM) as a
28 receptor for their entry to cells. In this study, a complete gene encoding SLAM of a
29 domestic cat was identified. The identity of feline SLAM with canine one was 73%,
30 and feline SLAM formed the same cluster with those of carnivores. Furthermore, feline
31 cell expressing feline SLAM supported growth of canine distemper virus (CDV) as
32 well as that expressing canine one. These results indicated that feline SLAM can
33 function as a receptor for morbilliviruses, and our established feline cells that express
34 feline SLAM might be useful for analysis of morbilliviruses originated from felids.
35
36 KEY WORDS: canine distemper virus, feline, signaling lymphocyte activation
37 molecule
2
38 Canine distemper virus (CDV) infects dogs and various species of carnivores.
39 It causes a fatal disease which involves the development of pyrexia, anorexia, nasal
40 discharge, conjunctivitis, diarrhea, leukopenia and encephalitis [2]. Recently, CDV was
41 recognized as a cause of morbidity and mortality in large felids, such as lions (Panthera
42 leo) in Tanzania’s Serengeti National Park [18]. It has also been found in lions, tigers
43 (Panthera tigris), leopards (Panthera pardus) and a jaguar (Panthera onca) in North
44 American zoos in 1991-1992 [4], one Siberian tiger (Panthera tigris altaica) in
45 Pokrovka, Russia, in 2004 [17] and tigers in a Japanese zoo in 2009 [14]. Although
46 CDV causes severe disease in various species, including large felids, it is still unclear
47 whether it can cause disease in domestic cats. In a serological survey of domestic cats,
48 some cats possessed virus-neutralizing antibody to CDV [9]. Furthermore, a feline
49 morbillivirus (FmoPV) was isolated from domestic cats in Hong Kong and might be
50 associated with tubulointerstitial nephritis [21]. Therefore, this suggests that domestic
51 cats can also be infected with morbilliviruses, including CDV.
52 Signaling lymphocyte activation molecule (SLAM) is expressed on activated T
53 and B cells, immature thymocytes, memory T cells [5, 7, 19], activated monocytes [12]
54 and mature dendritic cells [6, 11] and serves as a common receptor for morbillivirus [1,
55 20]. Canine SLAM expressed on cells of the immune system is also a major cellular
1
56 receptor for CDV infection [20]. However, a complete gene encoding SLAM of felids
57 including domestic cats has not been identified, and it is unclear whether it functions as
58 a cellular receptor for morbilliviruses, including CDV and FmoPV.
59 In this study, we identified the complete gene encoding feline SLAM and
60 established feline SLAM-expressing cells to assess whether feline SLAM functions as a
61 receptor for CDV.
62 To amplify the gene encoding feline SLAM, two primers, feline SLAM ATG
63 -1F (5’-TTC TCT TTC TTC AGT GGC TG-3’) and feline SLAM TGA-R (5‘-TCC CTT
64 CGC TGA GTC TCT G-3’), were designed based on the portion of the cat genome
65 homologous to the gene encoding canine SLAM. A feline thymus cDNA library of a
66 domestic cat (Bastard) was prepared using a cDNA Cycle® Kit according to the manual
67 (Invitrogen, Carlesbad, CA, U.S.A.). The gene encoding feline SLAM was amplified by
68 Takara Ex-Taq kit (Takara, Otsu, Japan). The reaction was carried out with the
69 following temperature profile: 94°C for 2 min, 40 cycles consisting of denaturation at
70 94°C for 30 sec, annealing at 60°C for 30 sec and extension at 72°C for 1 min and final
71 extension at 72°C for 10 min. The amplified products were purified by Minelute PCR
72 Purification kit (QIAGEN, Hilden, Germany). Sequencing reactions were performed
73 using six primers, feline SLAM ATG-1F, feline SLAM TGA-R, feline SLAM F
2
74 (5’-ATG AAC AGG TCT CCA CTC CG-3’), feline SLAM R (5’-CTG GAC TTG GGC
75 ATA GAT CG-3’), feline SLAM F1 (5’-TCA TTG GTG TCA TCT TGA CG-3’), and
76 feline SLAM R1 (5’-TGG GTC CAG TTC AAC ACC TT-3’) and a BigDye Terminator
77 v3.1 Cycle Sequencing kit (Applied Biosystems, Foster City, CA, U.S.A.). The reaction
78 was carried out with the following temperature profile: at 96°C for 1 min and 25 cycles
79 consisting of 96°C for 10 sec, 50°C for 5 sec, and 60°C for 4 min and nucleotide
80 sequences were analyzed by the Center of Gene Research at Yamaguchi University. The
81 nucleotide sequence was deposited in the DNA data bank of Japan (DDBJ) as accession
82 number AB771742.
83 Feline SLAM consisted of 1,014 bp encoding 337 amino acids. There was 73%
84 identity between the predicted amino acid sequences and canine SLAM. Feline SLAM
85 differed from canine SLAM at five (66, 67, 72, 74 and 82) of the twenty-one amino acid
86 residues at the SLAM interface which are involved in regulating the binding and
87 specificity of morbillivirus [16]. There was 78% identity between feline SLAM and the
88 pinniped SLAM of walrus (Odobenus rosmarus) and seals (Phoca larga). Phylogenetic
89 analysis showed that feline SLAM was closely related to those of various carnivores
90 (Fig. 1).
91 The gene encoding feline SLAM without the signal peptide and transmembrane
3
92 region was amplified from feline thymus cDNA by PCR with the primers,
93 pDis-Bgl-feline SLAM F (5’-TCA GAT CTG AGA GCT TGA TGA CTT GTC AA-3’)
94 and pDis-Sal-feline SLAM R (5’-TCG TCG ACG GGT CTT AGT TCT GGA GGA
95 T-3’). In order to amplify the corresponding canine gene, the gene encoding canine
96 SLAM without the signal peptide and transmembrane region was amplified from
97 pDisplay/cSLAM [15] with the primers, canineSLAM-95F (5’-CCA GAT CTG AGA
98 GCT TGA TGA ATT GCC C-3’) and CDV724R (5’-TCG TCG ACT CTT GGC ACC
99 GAA GAC TCT G-3’). PCR products were digested with restriction endonucleases
100 BglII and SalI and cloned into BglII and SalI sites of the expression plasmid pDisplay
101 (Invitrogen), which has genes for a signal peptide of mouse immunoglobulin
102 kappa-chain V-J2-C, a hemagglutinin A (HA) epitope tag upstream of multiple cloning
103 sites and neomycin-resistance. These expression plasmids were named
104 pDisplay/fSLAM(PDGFR) and pDisplay/cSLAM(PDGFR), respectively.
105 CRFK cells originating from feline kidney (ATCC Number: CCL-94) were
106 transfected with pDisplay/fSLAM(PDGFR) using LipofectamineTM LTX and PlusTM
107 Reagent (Invitrogen) and selected by the addition of 800 μg/ml of G418. The expression
108 plasmid pDisplay/cSLAM(PDGFR) and an empty plasmid pDisplay were also
109 transfected into CRFK cells. After selection with G418, SLAM expression was
4
110 confirmed by flow cytometric analysis using an anti-HA tagged monoclonal antibody
111 (clone HA-7; SIGMA-ALDRICH, Tokyo, Japan) or normal mouse IgG1 (R&D systems,
112 Minneapolis, MN, U.S.A.) as a negative control. Anti-normal mouse IgG labeled with
113 ALEXA Fluor 488 (Invitrogen) was used as a secondary antibody. The results showed
114 that feline and canine SLAM were expressed equally on the surface of CRFK cells
115 (47.23 and 47.16%, respectively) (Fig. 2A, B). These cells were named
116 CRFK/cSLAM(PDGFR), CRFK/fSLAM(PDGFR) and CRFK/pDisplay. For further
117 experiments, these cells were passaged less than three times.
118 Three CDV strains were used in this study. KDK-1 (genotype Asia-1) was
119 isolated from a diseased dog in Japan in 1991 using Vero cells [13] and propagated in
120 A72/cSLAM cells [15] after six passages in Vero cells. KochiO1A (genotype Asia-1)
121 was isolated from an oral swab of a dead masked palm civet in Kochi Prefecture in 2008
122 (manuscript in preparation) and was only propagated in A72/cSLAM cells. These field
123 isolates were not plaque-purified and were propagated less than six times in
124 A72/cSLAM cells. The Onderstepoort vaccine strain (genotype America-1) was only
125 propagated in Vero cells. Viral titers were determined by plaque assay using
126 CRFK/cSLAM [15] for KDK-1 and KochiO1A, and Vero cells (JCRB Number;
127 JCRB9013) for Onderstepoort [10]
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128 To analyze the function of these expressed SLAMs as receptors for CDV,
129 CRFK/fSLAM(PDGFR), CRFK/cSLAM(PDGFR) cells and CRFK/pDisplay were
130 infected with KDK-1, KochiO1A or Onderstepoort at a multiplicity of infection (MOI)
131 of 0.002, and cytopathic effect was observed. KDK-1 and KochiO1A produced large
132 syncytia in both CRFK/fSLAM(PDGFR) cells (Fig.2 D, G) and
133 CRFK/cSLAM(PDGFR) cells (Fig.2 E, H), but not in CRFK/pDisplay cells (Fig.2 F, I).
134 Onderstepoort produced syncytia in all cells (Fig.2 J-K).
135 CDV growth was compared in cells which were seeded on 6 well plates
136 (5.0×105 cells per well) and infected with KDK-1, KochiO1A and Onderstepoort at an
137 MOI of 0.002. After 1 hour adsorption, the inoculum was removed, the cells were
138 washed twice with DMEM without FCS, and then 5 ml of DMEM containing 10% FCS
139 was added. The supernatants were collected every 24 hr, and virus titers were
140 determined by plaque assay. The growth kinetics showed that KDK-1 and KochiO1A
141 efficiently replicated in both CRFK/cSLAM(PDGFR) and CRFK/fSLAM(PDGFR)
142 cells, but not in CRFK/pDisplay cells (Fig.3). The highest viral titers of KDK-1 were
143 8.8×105 in CRFK/fSLAM(PDGFR), 9.3×105 in CRFK/cSLAM(PDGFR) and 1.9×104
144 PFU/well in CRFK/pDisplay cells at 96 hr postinfection. The highest titers of
145 KochiO1A were 6.8×103 in CRFK/fSLAM(PDGFR) cells at 96 hr postinfection,
6
146 1.9×104 in CRFK/cSLAM(PDGFR) cells at 96 hr postinfection and 2.5×101 PFU/well in
147 CRFK/pDisplay cells at 72 hr postinfection. Compared to KochiO1A, KDK-1 replicated
148 well in CRFK/pDisplay cells. Since KDK-1 initially isolated using Vero cells, the strain
149 may also utilize alternative cellular receptor in addition to the SLAM. On the other hand,
150 Onderstepoort replicated more efficiently in CRFK/pDisplay cells than in
151 CRFK/fSLAM(PDGFR) or CRFK/cSLAM(PDGFR) cells. The highest viral titers of
152 Onderstepoort were 9.8×104 in CRFK/fSLAM(PDGFR) cells at 72 hr postinfection,
153 2.0×105 in CRFK/cSLAM(PDGFR) at 72 hr postinfection and 1.8×106 PFU/well, in
154 CRFK/pDisplay cells at 96 hr postinfection. Similar tendency in intracellular virus
155 growth was observed (data not shown).
156 In this study, the complete gene encoding feline SLAM was identified and
157 found to have higher homology with SLAM of other carnivores which are susceptible to
158 CDV infection rather than with SLAM of other animals susceptible to infection with
159 other morbilliviruses such as measles virus, rinderpest virus and peste-des-petits
160 ruminant virus. In addition, our established cell line, CRFK/fSLAM, supported
161 replication of KDK-1 and KochiO1A as well as CRFK/cSLAM, indicating that feline
162 SLAM, as well as canine SLAM, can serve as a receptor for CDV field strains.
163 Furthermore, Appel et al. [3] reported CDV replication in lymphatic tissues and
7
164 macrophages of experimentally infected cats without any clinical signs. These results
165 indicate that, in addition to canine lymphocytes, feline lymphocytes expressing SLAM
166 might also become infected with CDV.
167 Fatal infection of large felids with CDV has recently been reported worldwide.
168 In a Japanese safari-style zoo, 12 tigers developed respiratory and gastrointestinal
169 disease, and one tiger died of a neurological disorder following CDV infection [14].
170 One Siberian tiger presented symptoms of neurological signs and anorexia and
171 ultimately died in Russia [17]. Although SLAMs of large felids such as lions and tigers
172 have not been identified, SLAM from domestic cats belonging to the same family could
173 function as a receptor for CDV.
174 On the other hand, specific pathogen-free domestic cats inoculated with
175 homogenized tissues from a Chinese leopard (Panthera pardus japonensis) that had
176 died following natural infection with CDV did not show any clinical signs except for a
177 transient leukopenia [8], indicating that CDV was less pathogenic in domestic cats. Our
178 results show that CDV can use feline SLAM as a receptor. This is supported by the fact
179 that the CDV experimentally infected cats showed a tentative viremia associated with
180 CDV-infected PBMC [8]. Furthermore, in comparison with field strains, Onderstepoort
181 grew well in CRFK/pDisplay cells, but not in feline or canine SLAM-expressing cells.
8
182 This result is consistent with our previous observation [15], but the reason why
183 Onderstepoort cannot propagate well in SLAM-expressing cells remains unclear.
184 Further experiments will be required to clarify the mechanism behind this inhibition.
185 In 2012, FmoPV was isolated from CRFK cells [21] following subculture over
186 a prolonged period. On day 14 after the 8th passage, the cells displayed typical
187 cytopathic effects of rounding, followed by detachment from the monolayer, and on day
188 10 after the 16th passage, syncytia were observed [21]. Although SLAM is a common
189 receptor for morbilliviruses, including CDV, measles virus, rinderpest virus and
190 peste-des-petits ruminant virus, it is unclear whether FmoPV uses SLAM as a receptor.
191 However, this CRFK/fSLAM will be useful for isolation of morbilliviruses, including
192 CDV, from domestic cats.
193 In conclusion, feline SLAM was shown to serve as a receptor for CDV
194 infection.
195
196 ACKNOWLEDGMENTS 197 KDK-1 was kindly provided by Dr. Mochizuki (Kagoshima University). This 198 work was supported by grants from the Ministry of Education, Culture, Sports, Science, 199 and Technology of Japan, and Grant-in-Aid for Japan Society for the Promotion of 200 Science Fellows.
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276 Figure captions 277 Fig. 1. Phylogenetic relationship based on the amino acid sequence of SLAM. Feline 278 SLAM is shown in bold. Phylogenetic analysis was conducted using MEGA5. 279 280 Fig. 2. Expression of SLAM on the cell surface and development of syncytia after CDV 281 infection. (A-C) Flow cytometric analysis of SLAM expression on CRFK cells. CRFK 282 cells were transfected with expression plasmids and selected with G418. The resulting 283 cell lines were named CRFK/fSLAM(PDGFR) (A), CRFK/cSLAM(PDGFR) (B) and 284 CRFK/pDisplay (C). Cells were first incubated with anti-HA tagged monoclonal 285 antibody (clone HA-7) and then incubated with anti-normal mouse IgG labeled with 286 ALEXA Fluor 488. (D-K) CPE induced by CDV infection in CRFK/fSLAM(PDGFR) 287 (D, G, J), CRFK/cSLAM(PDGFR) (E, H, K) and CRFK/pDisplay cells (F, I, L). Cells 288 were infected with KDK-1 (D-F), KochiO1A (G-I) or Onderstepoort (J-K) strains of 289 CDV for 72 hr and then observed using microscopy. 290 291 Fig. 3. Growth kinetics of CDV in SLAM-expressing CRFK cells. KDK-1 (A), 292 KochiO1A (B) and Onderstepoort (C) infected into CRFK/fSLAM, CRFK/cSLAM and 293 CRFK/pDisplay cells at an MOI of 0.002. Mean values from two independent 294 experiments are shown. 295
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Figure 1
99 Cattle (AAK61860) 99 Zebu cattle (ABB58750) 100 Water buffalo (ABB58751) Goat (ABB58752) 99 98 Sheep (NP_001035378) 99 Pacific white-sided dolphin (BAH10670) 100 42 Killer whale (BAH10671) Pig (NP_001230749) Horse (XP 001504489) 96 Domestic cat 99 Spotted seal (BAH10672) 41 Walrus (BAH10673) 97 97 Giant panda (XP_002928483) 72 Mink (ACM90097) 75 37 Dog (NP_001003084) Raccoon dog (ACD47120) 100 Red fox (ACD47119) 94 Caribbean manatee (BAH10674) 100 African savanna elephant (XP_003415237) 100 Asiatic elephant (BAH10675) Small-eared galago (XP_003795232) 90 Cotton-top tamarin (AAG18445) 99 100 White-tufted-ear marmoset (XP_002760222) Bolivian squirrel monkey (XP_003938005) 100 Sumatran orangutan (XP_002809959) 100 Rhesus monkey (XP_001117605) 53 Human (AAK77968) 99 Chimpanzee (XP_513924) 62 Pygmy chimpanzee (XP_003811920) Rabbit (XP_002715271) House mouse (BAE96300) 100 Norway rat (NP_001102548)
0.05
1
Figure 2
A B C
47.23% 47.16% 0.96%
D E F
G H I
J K L
2
Figure 3
A B C 1.0.E+07 1.0.E+07 1.0.E+07 1.0.E+06 1.0.E+06 1.0.E+06
PFU) 1.0.E+05 PFU) 1.0.E+05 1.0.E+05 CRFK/fSLAM(P (PFU) DGFR) 1.0.E+04 1.0.E+04 1.0.E+04 CRFK/cSLAM(P 1.0.E+03 1.0.E+03 1.0.E+03 DGFR)
Virus titer ( titer Virus 1.0.E+02 ( titer Virus 1.0.E+02 titer Virus 1.0.E+02 CRFK/pDisplay 1.0.E+01 1.0.E+01 1.0.E+01 0 24 48 72 96 120144 0 24 48 72 96 120144 0 24 48 72 96 120144 Hours post infection Hours post infection Hours post infection
3