Identification of 2 novel type I IFN genes in Japanese flounder, Paralichthys olivaceus

Accepted Manuscript Identification of 2 novel type I IFN genes in Japanese flounder, Paralichthys olivaceus Yiwen Hu, Takaki Yoshikawa, Seangmin Chung, Ikuo Hirono, Hidehiro Kondo PII:

S1050-4648(17)30299-1

DOI:

10.1016/j.fsi.2017.05.054

Reference:

YFSIM 4607

To appear in:

Fish and Shellfish Immunology

Received Date: 21 March 2017 Revised Date:

17 May 2017

Accepted Date: 20 May 2017

Please cite this article as: Hu Y, Yoshikawa T, Chung S, Hirono I, Kondo H, Identification of 2 novel type I IFN genes in Japanese flounder, Paralichthys olivaceus, Fish and Shellfish Immunology (2017), doi: 10.1016/j.fsi.2017.05.054. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

ACCEPTED MANUSCRIPT

1

Identification of 2 novel type I IFN genes in Japanese flounder,

2

Paralichthys olivaceus

4

RI PT

3 Yiwen Hua,b, Takaki Yoshikawaa, Seangmin Chunga, Ikuo Hironoa and Hidehiro Kondoa

5 6

a

7

Science and Technology, Minato-ku, Tokyo, Japan

8

b

9

Marine Science and Technology, Zhejiang Ocean University, No. 1 of Haida Street,

SC

Laboratory of Genome Science, Graduate School of Tokyo University of Marine

M AN U

National Engineering Research Center of Marine Facilities Aquaculture, College of

Zhoushan, Zhejiang 316022, China

11

Corresponding author: Hidehiro Kondo

12

TEL: +81-3-5463-0174. FAX: +81-3-5463-0174.

13

E-mail: [email protected]

14

Address: Laboratory of genome science, Tokyo University of Marine Science and

15

Technology, Konan 4-5-7, Minato-ku, Tokyo 108-8477, Japan

17

EP

AC C

16

TE D

10

Keywords: Japanese flounder, Type I interferon, Cloning, PolyI:C

1

ACCEPTED MANUSCRIPT

18

Abstract Two novel type I interferon genes (JfIFN3 and JfIFN4) have been identified in

20

Japanese flounder Paralichthys olivaceus. Open reading frames of JfIFN3 and JfIFN4

21

were 555bp and 528bp, encoding 184aa and 175aa, respectively. The genomic

22

structures of JfIFN3 and JfIFN4 are composed of 5 exons and 4 introns. JfIFN4 has 2

23

conserved cysteine residues, while JfIFN3 has 4. JfIFN3 and JfIFN4 showed the highest

24

amino acid sequence identities to turbot IFN1 (74 %) and IFN2 (62 %), respectively.

25

Interestingly, JfIFN3 and JfIFN4 were clustered in distinct branches with JfIFN1 and

26

JfIFN2, which have reported so far. The mRNA levels of JfIFN4 were apparently

27

increased in the kidney and spleen at 3 hours after ployI:C injection, while JfIFN1-3

28

were not detected by RT-PCR.

SC

M AN U

TE D EP

30

AC C

29

RI PT

19

2

ACCEPTED MANUSCRIPT

31

1. Introduction The type I interferon (IFN) system plays a pivotal role in both innate and adaptive

33

immunity against virus in fish [1, 2]. Briefly, after dsRNA or ssRNA virus infection,

34

mostcells can recognize virus by the receptors and then express IFN [3]. The secreted

35

IFN bind to two IFN receptors, forming a ternary receptor complex and finally the

36

released signal which through JAK/STAT pathway induce expression of a wide range

37

of the IFN stimulated genes, some of which encode antiviral proteins [4, 5]. This

38

represents the antivirus innate immune response. Meanwhile, the secreted IFN can

39

stimulate dendritic cells, T cells and B cells, resulting in the production of specific

40

antibodies and cytotoxic T cells [5-8]. This represents the antivirus adaptive immune

41

response.

M AN U

SC

RI PT

32

In general, teleost fish type I interferons are divided into two groups depending on

43

the conserved cysteine residues in the mature peptide: group I and II IFNs containing

44

either two or four cysteines and result in forming one or two disulphide bonds,

45

respectively [9]. Phylogenetically, teleost type I IFNs can be further classified into six

46

subgroups, termed IFNa, b, c, d, e and f [10]. Recent study identified a novel subgroup

47

IFNh in large yellow croaker. Overall, a, d, e and h subgroups constitute group I IFNs

48

while b, c and f subgroups constitute group II IFNs [11].

AC C

EP

TE D

42

49

To date, a range of teleost species type I IFN genes have been identified [11-24],

50

where the gene copy number was found to vary among species. Salmonids possess the

51

most complex repertoire of type I IFN in fish [5]. There are eleven IFN genes that were

52

identified in Atlantic salmon initially and it is likely that there are more copies

3

ACCEPTED MANUSCRIPT

according to its recent genome data [25, 5]. Japanese flounder (Paralichthys olivaceus)

54

is an economically important marine fish in Japan, China and Korea [26]. Meanwhile, a

55

Japanese flounder IFN (JfIFN1) has been sequenced [27] and another IFN (JfIFN2) has

56

been deposited in the public database (AHB859752).

RI PT

53

Here we reported two novel IFN genes of Japanese flounder with complete open

58

reading frame cloned from spleen and kidney after polyI:C stimulation and named as

59

“JfIFN3” and “JfIFN4” tentatively. These two IFNs were differentially expressed

60

according to transcriptome data comparing fish samples with those injected with

61

polyI:C.

62 2. Materials and Methods

64

2.1. Treatments

TE D

63

M AN U

SC

57

Healthy Japanese flounder were kept at 20℃ and fed with commercial feed every

66

day. After acclimating for one week, each fish was intraperitoneally injected with 100μ

67

g/100μl polyI:C.

69 70

AC C

68

EP

65

2.2. cDNA cloning and structural analyses The partial JfIFN3 and JfIFN4 sequences were obtained from our RNA-sequence

71

data generated by NGS. Specific primers were designed for PCR to confirm their

72

coding sequences. The cDNA template for RT-PCR was synthesized from spleen and

73

kidney mRNA at 3 hours after polyI:C injection. PCR products were cloned into pGEM

74

T-easy vector (Promega, Japan) and sequenced. Furthermore, using the cDNA

4

ACCEPTED MANUSCRIPT

sequences obtained corresponding genomic sequences were searched for in the NCBI

76

database. The presence of signal peptide was predicted by using the SignalP 4.1 Server

77

(http://www.cbs.dtu.dk/services/SignalP/). Molecular weight and isoelectric point of the

78

amino

79

(http://web.expasy.org/compute_pi/). Multiple alignments of the amino acid sequences

80

were performed using Clustal Omega (http://www.ebi.ac.uk/Tools/msa/clustalo/).

81

Phylogenetic tree was constructed using Neighbor-Joining method.

sequences

determined

with

ExPASy

M AN U

82 83

were

SC

acid

RI PT

75

2.2 RT-PCR

Total RNA was extracted from spleen and kidney at 0, 3 and 24 hours after polyI:C

85

injection by RNAiso (Takara, Japan). Then 0.2 µg of RNA was used to synthesize

86

cDNA. Thermal cycling conditions were as follow; one cycle of 95℃ for 5 mins, 28

87

cycles of 95 ℃ for 30 s, 58 ℃ for 30 s, and 72 ℃ for 30 s, followed by one cycle of

88

72 ℃ for 5 mins. Genomic DNA from Japanese flounder muscle was used as a control.

EP

89

TE D

84

3. Results and discussion

91

3.1 cDNA cloning and characterization of Japanese flounder type I IFNs

92

AC C

90

The cDNA encoding JfIFN3 and JfIFN4 were determined (Accession numbers,

93

LC222627 and LC222628). By searching genomic sequence database using the

94

sequences, corresponding regions were deposited in the NCBI database as

95

NW_017860455 (from 2177 to 3402) and NW_017862086 (from 2403 to 3700),

96

respectively, and both genes have 5 exons and 4 introns. JfIFN3 and JfIFN4 cDNA

5

ACCEPTED MANUSCRIPT

contain open reading frames (ORF) of 555bp and 528bp, respectively. They encode

98

184aa and 175aa, and first 23aa and 16aa were predicted as signal peptides, respectively

99

(Fig. 1). Most IFNs possess a signal peptide depending on their deduced amino acid

100

sequences. Nevertheless, there are some reports on existing IFN transcript variants in

101

salmonids and cyprinids, which don’t have predicted signal peptides [12, 28, 29]. In this

102

study, all four JfIFNs were predicted to have a signal peptide. The molecular weights of

103

JfIFN3 and JfIFN4 mature peptides are 17.9kDa and 18.9kDa, with theoretical

104

isoelectric points of 5.7 and 10.1, respectively.

M AN U

SC

RI PT

97

The number of cysteine residues is one of characteristics to classify fish IFNs [9].

106

JfIFN4 has 2 conserved cysteine residues, while JfIFN3 has 4 (Fig. 1). Based on this,

107

JfIFN1, JfIFN2 and JfIFN4 belong to group I, while JfIFN3 belongs to group II. The

108

distinctive family signature motif of type I IFN conserved in the C-terminus of all

109

JfIFNs. JfIFN3 and JfIFN4 showed the highest identities with turbot IFN1 (74%) and

110

turbot IFN2 (62%), respectively. Phylogenetically, JfIFN3 and JfIFN4 were distinct

111

from JfIFN1 and JfIFN2 (Fig. 2).

113 114

EP

AC C

112

TE D

105

3.2. RT-PCR of Japanese flounder type I IFNs after polyI:C treatment All primers for JfIFNs were designed to span 2 introns to distinguish the PCR

115

products from genome and cDNA. All primer sets were able to amplify the genomic

116

DNA fragments. The mRNA of four JfIFNs were not detected in spleen and kidney

117

before stimulated with polyI:C by RT-PCR. At 3 hours after polyI:C injection, the

118

mRNA levels of JfIFN4 were increased both in kidney and spleen. However, the

6

ACCEPTED MANUSCRIPT

mRNAs of JfIFN1, JfIFN2 and JfIFN3 were not detected at 3 hours and 24 hours by

120

RT-PCR (Fig. 3.). It should be noted that JfIFN1 and 2 were not detected in our

121

preliminary NGS RNA-seq analyses in kidney and spleeen, while JfIFN3 was detected

122

faintly after polyI:C stimulation (data not shown).

RI PT

119

Previous study reported that JfIFN1 gene was constitutively expressed in healthy

124

tissues, including spleen and kidney. Moreover, JfIFN1 promoter could be enhanced in

125

HINAE cells by both extracellular and intracellular polyI:C stimulation [27]. It is

126

possible that JfIFN1 expression both in spleen and kidney are very low so we could not

127

detect JfIFN1 by RT-PCR in this study.

M AN U

SC

123

According to previous studies, there are three different pathways that can be

129

activated by RNA virus, and different IFN subgroups are associated with these specific

130

IFN induction pathways. In the case of polyI:C, which is a dsRNA virus analogue, be

131

recognized by MDA5, TLR3 and TLR22 in fish [5, 30-33]. On the other hand, ssRNA

132

virus may be recognized be RIG-I, TLR7 and TLR8 [5, 30, 31, 34, 35]. Early induction

133

of IFN after polyI:C stimuli was also reported in the other fish species [11, 20]. PolyI:C

134

and ssRNA virus differentially modulate the different IFN subgroups. In Atlantic

135

salmon, IFNa genes were strongly up-regulated by polyI:C, but slightly up-regulated by

136

TLR7 ligand S-27609, which is the analogue of imiquimod. IFNb genes were strongly

137

up-regulated by S-27607, but slightly up-regulated by polyI:C. IFNc genes were

138

sensitive to both polyI:C and S-27609 [36, 37]. JfIFN3, which may belong to subgroup

139

c, was not detected after polyI:C stimulation. This suggested that in the same subgroup

140

IFNs, induction pathway among different fish species may vary. In addition, different

AC C

EP

TE D

128

7

ACCEPTED MANUSCRIPT

IFN subgroups showed different expression patterns by polyI:C. In large yellow croaker,

142

upon polyI:C stimulation, the increase of IFNh transcripts was greatly higher than that

143

of IFNd in head kidney and spleen [11]. Thus, JfIFNs transcription appear to be also

144

regulated in a different pathway. Furthermore, JfIFN4 may play a more important role

145

in anti-dsRNA virus activity than JfIFN1, JfIFN2 and JfIFN3.

147

SC

146

RI PT

141

4. Conclusions

In summary, we identified two novel type I IFN genes (JfIFN3 and JfIFN4) in

149

Japanese flounder. These 2 molecules are likely to be orthologues of turbot IFN1 and

150

IFN2, rerspectively. Among the 4 IFNs, only mRNA levels of JfIFN4 were increased by

151

polyI:C injection, suggesting that the important role of JfIFN4 in anti-virus responses.

152

TE D

M AN U

148

Acknowledgement

154

This work was supported in part by grants from JSPS KAKENHI Grant Number

155

25292125.

AC C

156

EP

153

8

ACCEPTED MANUSCRIPT

157

References

158

[1] Robertsen, B., The interferon system of teleost fish. Fish Shellfish Immunol. 2006;

161 162 163

RI PT

160

20:172-191. [2] Zou, J., Secombes, CJ., Teleost fish interferons and their role in immunity. Dev. Comp. Immunol. 2011; 35:1376-1387.

[3] Yoneyama, M., Fujita, T., Recognition of viral nucleic acids in innate immunity. Rev.

SC

159

Med. Virol. 2010; 20: 4-22.

[4] Aggad, D., Mazel, M., Boudinot, P., Mogensen, K.E., Hamming, O.J., Hartmann, R.,

165

Kotenko, S., Herbomel, P., Lutfalla, G., Levraud, JP., The two groups of zebrafish

166

virus-induced interferons signal via distinct receptors with specific and shared

167

chains. J. Immunol. 2009; 183: 3924-3931.

TE D

168

M AN U

164

[5] Robertsen, B., The role of type I interferons in innate and adaptive immunity against viruses

in

Atlantic

salmon.

Dev.

170

http://dx.doi.org/10.1016/j.dci.2017.02.005.

Comp.

Immunol.

2017;

EP

169

[6] Le Bon, A., Thompson, C., Kamphuis, E., Durand, V., Rossmann, C., Kalinke, U.,

172

Tough, D.F., Cutting edge: enhancement of antibody responses through direct

173 174 175

AC C

171

stimulation of B and T cells by type I IFN. J. Immunol. 2006; 176: 2074-2078.

[7] Le Bon, A., Tough, D.F., Type I interferon as a stimulus for cross-priming. Cytokine & Growth Factor Rev. 2008; 19: 33-40.

176

[8] Longhi, M.P., Trumpfheller, C., Idoyaga, J., Caskey, M., Matos, I., Kluger, C.,

177

Salazar, A.M., Colonna, M., Steinman, R.M., Dendritic cells require a systemic

9

ACCEPTED MANUSCRIPT

178

type I interferon response to mature and induce CD4+ Th1 immunity with poly IC

179

as adjuvant. J. Exp. Med. 2009; 206: 1589-1602. [9] Zou, J., Tafalla, C., Truckle, J., and Secombes, CJ., Identification of a second group

181

of type I IFNs in fish sheds light on IFN evolution in vertebrates. J. Immunol. 2007;

182

179: 3859-3871.

RI PT

180

[10] Zou, J., Gorgoglione, B., Taylor, NG., Summathed, T., Lee, PT., Panigrahi, A.,

184

Genet, C., Chen, TY., UI, Hassan, M., Mughal, SM., Boudinot, P., Secombes, CJ.,

185

Salmonids have an extraordinary complex type I IFN system: characterization of

186

the IFN locus in rainbow trout Oncorhynchus mykiss reveals two novel IFN

187

subgroups. J. Immunol. 2014; 193:2273-2286.

M AN U

SC

183

[11] Ding, Y., Ao, J., Huang, X. and Chen, X., Identification of two subgroups of type I

189

IFNs in Perciforme fish large yellow croaker Larimichthys crocea provides novel

190

insights into function and regulation of fish type I IFNs. Front. Immunol. 2016;

191

7:343.

EP

TE D

188

[12] Robertsen, B., Bergan, V., Rokenes, T., Larsen, R., Albuquerque A., Atlantic

193

salmon interferon genes: cloning, sequence analysis, expression, and biological

194

AC C

192

activity. J Interferon Cytokine Res. 2003; 23:601-612.

195

[13] Altmann, SM., Mellon, MT., Distel, DL., Kim, CH., Molecular and functional

196

analysis of an interferon gene from the zebrafish, Danio rerio. J. Virol. 2003;

197

77:1992-2002.

198 199

[14] Lutfalla, G., Crollius, HR., Stange-Thomann, N., Jaillon, O., Mogensen, K., Monneron,

D.,

Comparative

10

genomic

analysis

reveals

ACCEPTED MANUSCRIPT

independent expansion of a lineage-specific gene family in vertebrates: the class II

201

cytokine receptors and their ligands in mammals and fish. BMC Genomics 2003;

202

4:29.

RI PT

200

[15] Zou, J., Yoshiura, Y., Dijkstra, JM., Sakai, M., Ototake, M., Secombes, C.,

204

Identification of an interferon gamma homologue in fugu, Takifugu rubripes. Fish

205

Shellfish Immunol. 2004; 17:403-409.

SC

203

[16] Long, S., Milev-Milovanovic, I., Wilson, M., Bengten, E., Clem, LW., Miller, NW.,

207

Chinchar, VG., Identification of a cDNA encoding channel catfish interferon. Dev.

208

Comp. Immunol. 2004; 28:97-111.

M AN U

206

[17] Zhang, YB., Jiang, J., Chen, YD., Zhu, R., Shi, Y., Zhang, QY., Gui, JF., The innate

210

immune response to grass carp hemorrhagic virus (GCHM) in the cultured

211

Carassius auratus blastulae (CAB) cells. Dev. Comp. Immunol. 2007; 31: 232-243.

TE D

209

[18] Kitao, Y., Kono, T., Korenaga, H., Iizasa, T., Nakamura, RS., Sakai, M.,

213

Characterzation and expression analysis of type I interferon in common carp

214

Cyprinus carpio L. Mol. Immunol. 2009; 46: 2548-2556.

216 217 218

[19] Casani, D., Randelli, E., Costantini, S., Facchiano, AM., Zou, J., Martin, S.,

AC C

215

EP

212

Secombes, CJ., Scapigliati, G., Buonocore, F., Giuseppe S., Francesco B., Molecular characterization and structural analysis of an interferon homologue in

sea bass (Dicentrarchus labrax L.) Mol. Immunol. 2009; 46: 943-952.

219

[20] Wan, Q., Wicramaarachchi, WD., Whang, I., Lim, BS., Oh, MJ., Jung, SJ., Kim,

220

HC., Yeo, SY., Lee, J. Molecular cloning and functional characterization of two

221

duplicated two-cysteine containing type I interferon genes in rock bream

11

ACCEPTED MANUSCRIPT

222

Oplegnathus fasciatus. Fish Shellfish Immunol. 2012; 33: 886-898. [21] Pereiro, P., Costa MM., Díaz-Rosales P., Dios S., Figueras A., Novoa B., The first

224

characterization of two type I interferons in turbot (Scophthalmus maximus) reveals

225

their differential role, expression pattern and gene induction. Dev. Comp. Immunol.

226

2014; 45: 233-244.

RI PT

223

[22] Wan, Q., Wicramaarachchi, WD., Whang, I., Lim, BS., Oh, MJ., Jung, SJ., Kim,

228

HC., Yeo, SY., Lee, J., Molecular cloning and functional characterization of two

229

duplicated two-cysteine containing type I interferon genes in rock bream

230

Oplegnathus fasciatus. Fish Shellfish Immunol. 2012; 33:886-898.

M AN U

SC

227

[23] Ohta, T., Ueda, Y., Ito, K., Miura, C., Yamashita, H., Miura, T., Tozawa, Y.,

232

Anti-viral effects of interferon administration on sevenband grouper, Epinephelus

233

septemfasciatus. Fish Shellfish Immunol. 2011; 30: 1064-1071.

TE D

231

[24] Chen, YM., Kuo, CE., Chen, GR., Kao, YT., Zou, J., Secombes, CJ., Chen, TY.,

235

Functional analysis of an orange-spotted grouper (Epinephelus coioides) interferon

236

gene and characterisation of its expression in response to nodavirus infection. Dev.

237

Comp. Immunol. 2014; 46: 117-128.

AC C

EP

234

238

[25] Sun, B., Robertsen, B., Wang, Z., Liu, B., Identification of an Atlantic salmon IFN

239

multigene cluster encoding three IFN subtypes with very different expression

240 241 242

properties. Dev. Comp. Immunol. 2009; 33: 547-558. [26] Seikai, T., Flounder culture and its challenges in Asia. Rev. Fish Sci. 2002; 10: 421-432.

12

ACCEPTED MANUSCRIPT

[27] Ohtani, M., Hikima, J., Hwang, SD., Morita, T., Suzuki, Y., Kato, G., Kondo, H.,

244

Hirono, I., Jung, TS., Aoki, T., Transcriptional regulation of type I interferon gene

245

expression by interferon regulatory factor-3 in Japanese flounder, Paralichthys

246

olivaceus. Dev Comp Immunol. 2012; 36: 697-706.

RI PT

243

[28] Long, S., Milev-Milovanovic, I., Wilson, M., Bengten, E., Clem, L.W., Miller,

248

N.W., Chinchar, V.G., Identification and expression analysis of cDNAs encoding

249

channel catfish type I interferons. Fish Shellfish Immunol. 2006; 21: 42-59.

M AN U

SC

247

[29] Purcell, M.K., Laing, K.J., Woodson, J.C., Thorgaard, G.H., Hansen, J.D.,

251

Characterization of the interferon genes in homozygous rainbow trout reveals two

252

novel genes, alternate splicing and differential regulation of duplicated genes. Fish

253

Shellfish Immunol. 2009; 26: 293-304.

TE D

250

[30] Bergan, V., Kileng, O., Sun, B., Robertsen, B., Regulation and function of

255

interferon regulatory factors of Atlantic salmon. Mol. Immunol. 2010; 47:

256

2005-2014.

EP

254

[31] Lauksund, S., Svingerud, T., Bergan, V., Robertsen, B., Atlantic salmon IPS-1

258

mediates induction of IFNa1 and activation of NF-kappaB and localizes to

259

AC C

257

mitochondria. Dev. Comp. Immunol. 2009; 33: 1196-1204.

260

[32] Matsuo, A., Oshiumi, H., Tsujita, T., Mitani, H., Kasai, H., Yoshimizu, M.,

261

Matsumoto, M., Seya, T., Teleost TLR22 recognizes RNA duplex to induce IFN

262

and protect cells from birnaviruses. J. Immunol. 2008; 181: 3474-3485.

263

[33] Vidal, R., Gonzalez, R., Gil, F., Characterization and expression analysis of Toll

13

ACCEPTED MANUSCRIPT

264

like receptor 3 cDNA from Atlantic salmon (Salmo salar). Genet. Mol. Res. 2015;

265

14: 6073-6083. [34] Lee, P.T., Zou, J., Holland, J.W., Martin, S.A., Kanellos, T., Secombes, C.J.,

267

Identification and characterization of TLR7, TLR8a2, TLR8b1 and TLR8b2 genes

268

in Atlantic salmon (Salmo salar). Dev. Comp. Immunol. 2013; 41: 295-305.

RI PT

266

[35] Heil, F., Hemmi, H., Hochrein, H., Ampenberger, F., Kirschning, C., Akira, S.,

270

Lipford, G., Wagner, H., Bauer, S., Species-specific recognition of single-stranded

271

RNA via toll-like receptor 7 and 8. Science. 2004; 303: 1526-1529.

M AN U

SC

269

272

[36] Kileng, Φ., Albuquerque, A., Robertsen, B., Induction of interferon system genes

273

in Atlantic salmon by the imidazoquinoline S-27609, a ligand for Toll-like receptor

274

7. Fish Shellfish Immunol. 2008; 24: 514-522.

[37] Sun, B., Robertsen, B., Wang, Z., Liu, B., Identification of an Atlantic salmon IFN

276

multigene cluster encoding three IFN subtypes with very different expression

277

properties, Dev. Comp. Immunol. 2009; 33: 547-558.

EP AC C

278

TE D

275

14

ACCEPTED MANUSCRIPT

Legends for figures

280

Fig. 1. Multiple alignment of JfIFNs with turbot type I IFNs by using Clustal Omega.

281

jfIFN, Japanese flounder IFN (jfIFN1, BAH84776; jfIFN2, AHB59752). smIFN,

282

Scophthalmus maximus IFN (smIFN1, AID59461; smIFN2, AID59462). The

283

sequences with underlines indicate putative signal peptides. The conserved

284

cysteines positions are colored with red and the distinctive family signature motif

285

of type I IFN are highlighted with grey.

SC

RI PT

279

Fig. 2. Phylogenetic tree of JfIFNs and other fish IFNs. Genebank accession numbers of

287

the selected fish IFN sequences for analysis: ssIFN, Salmo salar IFN (ssIFNa1,

288

ACE75690; ssIFNa2, NP001117042; ssIFNa3, ACE75687; ssIFNb1, ACE75691;

289

ssIFNb2, ACE75693; ssIFNb3, ACE75689; ssIFNc1, ACE75692; ssIFNc2,

290

ACE75694; ssIFNc3, ACE75688; ssIFNd, AFV08801). omIFN, Oncorhynchus

291

mykiss IFN (omIFNa1, CCP42398; omIFNa3, CCV17397; omIFNa4, CCV17398;

292

omIFNb3, CCV17399; omIFNb4, CCV17400; omIFNb5, CCV17401; omIFNc1,

293

CCV17402; omIFNc2, CCV17403; omIFNc3, CCV17404; omIFNc4, CCV17405;

294

omIFNe1, CCV17406; omIFNe2, CCV17407; omIFNe3, CCV17408; omIFNe4,

296

TE D

EP

AC C

295

M AN U

286

CCV17409; omIFNe5, CCV17410; omIFNe6, CCV174011; omIFNe7, CCV17412;

omIFNf1, CCV17413; omIFNf2, CCV17414). smIFN, Scophthalmus maximus IFN

297

(smIFN1, AID59461; smIFN2, AID59462). drIFN, Danio rerio IFN (drIFNp1,

298

NP997523;

299

NP001155212). lcIFN, Larimichthys crocea IFN (lcIFNd, API68651; lcIFNh,

300

API68650). olIFN, Oryzias Latipes IFN (olIFNa, BAU25608; olIFNd, BAU25609).

drIFNp2,

NP001104552;

15

drIFNp3,

NP001104553;

drIFNp4,

ACCEPTED MANUSCRIPT

301

gaIFN, Gasterosteus aculeatus IFN (gaIFN1, CAM31706; gaIFN2, CAM31707;

302

gaIFN3, CAM31708). ccIFN, Cyprinus carpio IFN, BAG68522. Fig. 3. Expression analysis of IFN genes in kidney and spleen stimulated with polyI:C

304

by RT-PCR. β-actin gene was used as an internal control. Genomic DNA from

305

Japanese flounder muscle was used as a control to compare with the target size.

AC C

EP

TE D

M AN U

SC

RI PT

303

16

ACCEPTED MANUSCRIPT

Table. 1 List of the primers used in this study

primer sequence (5 '-3 ')

Cloning PCR JfIFN3 F

ATGATGATGCCCTCTTCAGTCA

Cloning PCR JfIFN3 R

TCATGTGAAACAGCTGTGGTG

Cloning PCR JfIFN4 F

ATGATCAGGTGCACCATCATC

Cloning PCR JfIFN4 R

CTAGCGCCTCCTGCTGGC

RT-PCR JfIFN1 F

CCTGTTTGAGGAGGATTCCA

RT-PCR JfIFN1 R

CATGGCGTGACAGTCTCTTG

RT-PCR JfIFN2 F

CTGGAGGAGGTGGTGTTGTT

RT-PCR JfIFN2 R

TTCTTCTTGTGGCCGTCACT

RT-PCR JfIFN3 F

GCCAGGTATTATGGGTGGTG

RT-PCR JfIFN3 R

GCTGCTGCAAAACATCTGTC

RT-PCR JfIFN4 F

TGACCTCTATCGCCATGACA

RT-PCR JfIFN4 R

GCGGTCCAGAGTCCTTCTAA

RT-PCR β-actin F

TGATGAAGCCCAGAGCAAGA

RT-PCR β-actin R

CTCCATGTCATCCCAGTTGGT

AC C

EP

TE D

M AN U

SC

RI PT

Primer

ACCEPTED MANUSCRIPT

AC C

EP

TE D

M AN U

SC

RI PT

Fig. 1

ACCEPTED MANUSCRIPT

AC C

EP

TE D

M AN U

SC

RI PT

Fig. 2

ACCEPTED MANUSCRIPT

AC C

EP

TE D

M AN U

SC

RI PT

Fig. 3.

ACCEPTED MANUSCRIPT

Highlights Two novel interferon genes were identified in Japanese flounder.

•

The phylogenetic relationships were evaluated.

•

One of the interferon genes was induced after polyI:C treatment.

AC C

EP

TE D

M AN U

SC

RI PT

•