Identification and Expression of the Laboratory of Genetics and Physiology 2 Gene in Common Carp Cyprinus Carpio

Journal of Fish Biology (2014) doi:10.1111/jfb.12541, available online at wileyonlinelibrary.com

Identification and expression of the laboratory of genetics and physiology 2 gene in common carp Cyprinus carpio

X. L. Cao*†, J. J. Chen†,Y.Cao†,G.X.Nie†,Q.Y.Wan*,L.F.Wang† and J. G. Su*‡

*College of Animal Science and Technology, Northwest A&F University, Yangling 712100, People’s Republic of China and †College of Fisheries, Henan Normal University, Xinxiang 453007, People’s Republic of China

(Received 29 April 2014, Accepted 9 September 2014)

In this study, a laboratory of genetics and physiology 2 gene (lgp2) from common carp Cyprinus carpio was isolated and characterized. The full-length complementary (c)DNA of lgp2 was 3061 bp and encoded a polypeptide of 680 amino acids, with an estimated molecular mass of 77 341⋅2Daanda predicted isoelectric point of 6⋅53. The predicted protein included four main overlapping structural domains: a conserved restriction domain of bacterial type III restriction enzyme, a DEAD–DEAH box helicase domain, a helicase super family C-terminal domain and a regulatory domain. Real-time quantitative polymerase chain reaction (PCR) showed widespread expression of lgp2, mitochondrial antiviral signalling protein (mavs) and interferon transcription factor 3 (irf3) in tissues of nine organs. lgp2, mavs and irf3 expression levels were significantly induced in all examined organs by infection with koi herpesvirus (KHV). lgp2, mavs and irf3 messenger (m)RNA levels were significantly up-regulated in vivo after KHV infection, and lgp2 transcripts were also significantly enhanced in vitro after stimulation with synthetic, double-stranded RNA polyinosinic polycytidylic [poly(I:C)]. These findings suggest that lgp2 is an inducible protein involved in the innate immune defence against KHV in C. carpio. These results provide the basis for further research into the role and mechanisms of lgp2 in fishes. © 2014 The Fisheries Society of the British Isles

Key words: gene cloning; KHV; lgp2; mRNA expression.

INTRODUCTION Common carp Cyprinus carpio L. 1758 is one of the most important aquaculture fish species worldwide. Viral infections including koi C. carpio herpesvirus (KHV) have recently become a major problem in the C. carpio aquaculture industry. KHV is known to cause gill and skin damage in koi and common carp. The disease was first recognized in Israel and the U.S.A. (Hedricka et al., 2000), and has since has been reported in Europe, including Germany (Bretzinger et al., 1999) and the U.K. (Gilad et al., 2003). In Asia, KHV has been reported in Indonesia (Rukyani, 2002), Japan (Sano et al., 2002) and Taiwan (Tu et al., 2004) and is believed to have been imported into China. Rapid spread of the disease is associated with the import and export of ornamen- tal fish. KHV is a linear, double-stranded (ds) DNA virus with a genome of125–290

‡Author to whom correspondence should be addressed. Tel.: +86 29 87092139; email: [email protected] 1

kbp contained within a T = 16 icosahedral capsid and surrounded by a proteinaceous matrix (the tegument) and a lipid envelope containing membrane-associated proteins (Zhou et al., 2000). A better understanding of the immune response of C. carpio against this virus is essential to its prevention and the development of a healthy aquaculture industry. The recognition of viruses by host cells is mediated by pathogen recognition receptors that sense virus-specific pathogen-associated molecular patterns, typically viral nucleic acids. The nucleic acid sensing transmembrane toll-like receptors (TLR), tlr3, 7, 8, and 9, localize to intracellular compartments and have well-known ligand specificities: tlr3 detects dsRNA, tlr7 and tlr8 detect single-stranded (ss) RNA and tlr9 responds to patterns found in microbial DNA and oligodeoxynucleotides encoding unmethylated CpG motifs (Takeda & Akira, 2007). In contrast, cytosolic RNA ligands are recognized by the retinoic acid-inducible gene-1-like receptor (RLR, rig-I) and melanoma differentiation-associated gene 5 (mda5) (Yoneyama et al., 2004, 2005). rig-I and mda5 are characterized by a core DExH box domain fused to tandem N-terminal caspase activation and recruitment domain (CARD) motifs that are essential for propagating downstream signal transduction. A third RLR-related gene, laboratory of genetics and physiology 2 (lgp2), shows high sequence similarity to mda5 and rig-I DExH box domains but lacks N-terminal CARD homology. Expression of lgp2 from a plasmid vector negatively regulates interferon (ifn) production and antiviral signalling (Rothenfusser et al., 2005; Yoneyama et al., 2005; Komuro & Horvath, 2006; Saito et al., 2007), but analyses using mice deficient in Lgp2 indicated disparate functions of Lgp2 in response to different viruses (Venkataraman et al., 2007). Recent evidence suggests that Lgp2 acts as a positive regulator of Ifn responses to diverse viruses and may act in concert with mda5 and rig-I (Saito et al., 2007). Although the structure and function of lgp2 have been described, its role in the response to DNA viruses is currently unknown. Its molecular description and expression profile have not been reported in C. carpio. In this study, the C. carpio lgp2 gene was cloned and assayed and its tissue distribution and downstream associated gene mRNA expression profiles (including mavs and irf3 mRNAs, GenBank accession numbers: HQ850440.1 and HQ850443.1) analysed following KHV challenge. The lgp2 mRNA time-dependent expression in epithelioma papulosum cyprini (EPC) cells after polyinosinic polycytidylic [poly(I:C)] stimulation or KHV infection was also examined. The results of this study will provide the basis for understanding the function of lgp2 in cellular responses to dsDNA viruses, and will expand knowledge regarding innate immune mechanisms in teleosts.

MATERIALS AND METHODS

FISH, VIRUS DETECTION, IMMUNE CHALLENGE AND SAMPLE COLLECTION Nine month-old C. carpio were supplied by the Henan Institute of Aquaculture, Henan, China. They were maintained in indoor tanks equipped with a recirculating water system at a water temperature of 25∘ C for 7 days prior to experimentation. The fish were handled according to the guidelines of the China Law for Animal Health Protection and Instructions for Granting Permits for Animal Experimentation for Scientific Purposes [ethics approval number: SCXK (YU) 2005-0001].

The putative KHV outbreak was found when diseased fish showed clinical signs including lethargy, swimming at the water surface, dark or reddened skin, sandpaper-like skin lesions and deep-red gills with pale and white patches. KHV was confirmed by polymerase chain reaction (PCR)-based methods. DNA was extracted from gill, spleen and kidney of infected fish using DNAzol (Invitrogen; www.lifetechnologies.com), according to the manufacturer’s protocols. The specific primer set, HV-F (forward) and HV-R (reverse), developed byGray et al. (2002) was used to amplify a 290 bp fragment. DNA amplification was carried out according to the Manual of Diagnostic Tests for Aquatic Animals (Anon., 2009). The PCR product was subjected to electrophoresis, and the results were analysed by gel imaging and analysis. Cyprinus carpio showing various degrees of gill damage were used for virus isolation. Gill, kidney and spleen of diseased fish were removed aseptically and pooled, the pooled tissue was ground into a smooth paste in a cold sterile mortar, and the resultant homogenate was resus- pended in 9 ml of Hank’s balanced salt solution (HBSS, Gibco; www.lifetechnologies.com) supplemented with 2% foetal bovine serum (FBS). The tissue suspension was centrifuged (1500 g)at4∘ C for 15 min to pellet tissue debris. The clarified supernatant was diluted 1:5 (v/v) with supplemented HBSS and filtered through a⋅ 0 45 μm membrane filter to remove any bacterial contamination. The filtrate was stored at∘ 4 C for KHV challenge. For viral infection, C. carpio weighing 100–150 g were injected intraperitoneally with 4 250–300 μl of KHV at a dose of 10 tissue culture infective dose50 per g body mass. A control group was injected with an equal amount of saline. At 72 h post-infection (hpi), reverse transcription (RT)-PCR and nested PCR were performed to confirm KHV infection. Fish were anaesthetized with 100 mg l−1 of MS-222 and dissected, and tissue samples were collected at 0, 24, 48 and 72 hpi (three C. carpio for each time interval) for total RNA isolation.

RNA EXTRACTION AND CDNA SYNTHESIS Total RNA extraction was performed using Triazol reagent, according to the manufacturer’s instructions (TaKaRa; www.takara-bio.com). The quality and concentration of the total RNA were determined by gel electrophoresis and ultraviolet spectrophotometry. Three micrograms of total RNA was incubated with RNase-free DNase I (MBI Fermen- tas; www.thermoscientificbio.com) to remove genomic DNA and then reverse transcribed into cDNA using Oligo (dT) 20 primer and MMLV reverse transcriptase, according to the manufacturer’s instructions (TaKaRa).

GENE CLONING AND SEQUENCING The lgp2 cDNA sequence from C. carpio was cloned using degenerate primers designed based on multiple alignments with DHX58 in zebra fish Danio rerio (Hamilton 1822) (accession number: NM-001257157.1), channel catfish Ictalurus punctatus (Rafinesque 1818) (accession number: JQ008941.1), Japanese flounder Paralichthys olivaceus (Temminck & Schlegel 1846) (accession number: HM070372.1), grass carp Ctenopharyngodon idella (Valenciennes 1844) (accession number: FJ813483.2), Atlantic salmon Salmo salar L. 1758 (accession number: BT045378.1) and goldfish Carassius auratus (L. 1758) (accession number: JF970227.1). PCR was performed with the degenerate primers LGP2F (forward) and LGP2R (reverse) (Table I), using the cDNA generated from C. carpio spleen. The PCR programme was one cycle at 94∘ C for 3 min; 35 cycles at 94∘ C for 30 s, 58∘ C for 30 s and 72∘ C for 1 min and one cycle at 72∘ C for 10 min. PCR products were then purified from agarose gels using a DNA Gel Extrac- tion Kit (Axygen Scientific, Inc.; www.axygen.com). The purified DNA fragments were ligated into pDM19-Tvectors (TaKaRa), transformed into competent Escherichia coli DH5a cells and incubated on Luria–Bertani (LB) agar plates. Positive colonies were screened by colony PCR and sent to a commercial company (Beijing Genomics Institute; www.economicexpert.com) for sequencing. Full-length cDNA sequences of C. carpio lgp2 were acquired by rapid amplification of cDNA end (RACE) using the 5′ and 3′ full RACE Kit with TAP (TaKaRa), according to the manufacturer’s instructions. To obtain the unknown 3′ region, primer pairs LGP2-3-inner and LGP2-3-outer (Table I) were used for primary PCR and nested PCR, respectively. The amplified PCR product was cloned and sequenced as described above. Similarly, the5′ end

Table I. Primer sequences used in this study

Amplicon length (nt) and primer information Primer name Sequence(5′-3′) lgp2 LGP2F (forward) CATHATMTGGCTKCCYAC Gene clone LGP2R (reverse) AAGGCRTCCABCATDCGSAC 855 bp LGP2-3-inner GGCAATACAACGATGCTCTTCTC 3′ RACE LGP2-3-outer ACCACTTGAAGGCAATGATGTT 2133 bp L5-inner CCAGGTGTTTCTTGGTGATGTA 5′ RACE L5-outer GTGACATTCGTCTATTACCA 254 bp 3′ RACE outer primer TACCGTCGTTCCACTAGTGATTT 3′ RACE inner primer CGCGGATCCTCCACTAGTGATTTCA CTATAGG 5′ RACE outer primer CATGGCTACATGCTGACAGCCTA 5′ RACE inner primer CGCGGATCCACAGCCTACTGATGA TCAGTCGATG CF (forward) GCCTGACGACGTGCTGGAC Confirming sequence CR (reverse) CTAGATGTGATATACAGACTCAT 2912 EF1𝛼-F GTGCCGTGCTGATTGTTGCT ef1𝛼 EF1𝛼-R AAAGCCAGGAGGGCGTGTT 91 bp HV-F (forward) GACACCACATCTGCAAGGAG KHV HV-R (reverse) GACACATGTTACAATGGTCGC 292 bp q-PCR 𝛽-actin-F GATGATGAAATTGCCGCACTG 𝛽-actin 𝛽-actin-R ACCAACCATGACACCCTGATGT 151 bp L-qF CACAGCCAATGCCAAAGTAG LqR GCAGGGTAAAGTCAGTCAGC I-qF CTGTTGGGTTGCCAGTTTTC I-qR CAGCATTGTATGGAGGGTCT IRF-32F CAGGCATACGGAGGACATT IRF-32R TGGCTTCAGGTCTGTTTTTG

Note that H = A/C/T, M = A/C, K = G/T, Y = C/T, R = A/G, B = C/G/T, D = A/G /T, S = C/G. KHV, koi herpesvirus; RACE, rapid amplification of cDNA end; q-PCR, real-time polymerase chain reaction. of lgp2 was obtained by nested PCR using primer pairs L5-inner and L5-outer (Table I). The full-length cDNA sequence was confirmed by sequencing the PCR product amplified byCF (forward) and CR (reverse) primers (Table I) within the predicted 5′ and 3′ untranslated regions (UTR), respectively.

SEQUENCE ANALYSES Sequence homology was determined using the basic local alignment search tool (BLAST) programme (www.ncbi.nlm.nih.gov/blast) and matrix global alignment tool (MatGAT) (http://Bitincka.com/ledion/matgat/). Protein structure was predicted using the expert protein analysis system (www.expasy.org) and sequence manipulation suite programmes (www.bioinformatics.org/sms). Protein domain features were predicted using the simple modular architecture research tool (SMART) (http://smart.embl-heidelberg.de/), Pfam database search (http://pfam.sanger.ac.uk/search/) and putative conserved domain database (Finn et al., 2008). Intra-domain features were predicted by scanning the sequence against the PROSITE

database (http://us.expasy.org/tools/scanprosite/). Multiple alignment of the full-length or partial protein sequences of known or predicted Rig-I, Mda5 and Lgp2 was generated by CLUSTALW (www.clustal.org) and used for construction of a phylogenetic tree using the neighbour-joining method within the Mega 4.0.2 programme with C. carpio Tlr3 sequence as an outgroup.

REAL-TIME QUANTITATIVE PCR Real-time quantitative PCR (q-PCR) was conducted using an ABI PRISM 7500 Sequence Detector System (PerkinElmer Applied Biosystems; http://appliedbiosystems.com.cn/) with SYBR Green Master mix (TaKaRa). The housekeeping gene 𝛽-actin served as an internal reference gene, using the gene-specific primers 𝛽-actin-F (forward) and 𝛽-actin-R (reverse) (Table I). Gene-specific primers (Table I) for q-PCR were designed for single, gene-specific amplification of nucleotides. The q-PCR mixture consisted of2 μl of cDNA sample, 7⋅6 μlof nuclease-free water, 10 μlof2× SYBR Green PCR master mix (TaKaRa) and 0⋅2 μl of each gene-specific primer (10 mM). The PCR cycling conditions were one cycle at95∘ C for 30 s; 40 cycles at 95∘ Cfor5sand60∘ C for 30 s; one cycle at 95∘ C for 15 s, 60∘ C for 30 s and 95∘ C for 15 s; followed by dissociation curve analysis (65–95∘ C at increments of 0⋅5∘ Cfor 5 s) to verify the amplification of a single product. The relative expression ratios of the target genes in the treated group v. the control group were calculated by the 2−ΔΔCT method (Livak & Schmittgen, 2001). The expression data obtained from independent biological replicates were subjected to one-way analysis of variance (ANOVA), followed by unpaired, two-tailed t-tests. Values of P < 0⋅05 were considered statistically significant.

CELL CULTURE AND TREATMENT Cyprinus carpio EPC cells were grown in Dulbecco’s modified Eagle medium: nutrient mixture F12 (DMEM-F12) medium supplemented with 10% inactivated FBS (Gibco BRL), 100 U ml−1 of penicillin and 100 U ml−1 of streptomycin sulphate. Cells were maintained at 28∘ C in six-well tissue culture plates. All stimulations and infections were conducted in four paral- lel wells. For synthetic dsRNA stimulation, poly(I:C) (Sigma Aldrich; www.sigmaaldrich.com) dissolved in phosphate-buffered saline (PBS) was heated to 55∘ C for 5 min and allowed to cool to room temperature. The cells (1 × 106) were treated with 5 mg ml−1 (terminal concentration) of poly(I:C) or PBS (control). For kinetic studies, cells were harvested at 0, 2, 8 and 24 h post-stimulation, and RNA was isolated and reverse transcribed. For virus infection, EPC cells were infected with KHV at a multiplicity of infection of 1. Control cells were treated with PBS. To determine the time-dependent expression profiles, cells were harvested at 0, 2, 8, 16, 24and 48 hpi, and RNA was extracted and reverse transcribed.

RESULTS

CLONING AND SEQUENCE ANALYSES The first fragments of lgp2 were amplified using degenerate primers (Table I). Gene-specific primers (Table I) were designed based on these sequences andused for RACE to produce full-length cDNA of the target genes. The full-length cDNA of lgp2 was identified as a 3061 nucleotide cDNA sequence with an open reading frame encoding a putative protein of 680 aa (Fig. 1), a 5′ UTR of 126 nucleotides and a 3′ UTR of 895 nucleotides, including a poly(A) tail. Analysis of conserved domains revealed the presence of a DEAD–DEAH box helicase (DExD-H) domain, a helicase super family C-terminal (HELICc) domain and a regulatory domain (RD) at the C-terminus.

1 GCACGGGCCGCGCTGGTCACGCTGCCGGCAAGATTCGCGGGGCACGCGCAGGATGTCGCG 61 ATCCGGGTCGCCGAATTCGCGCCTGACGACGTGCTGGACGAACTGCAGATCGAAGATCCG 121 TTCGAAAATGACCGGACTATACGACGGCATCCCGGTGACCGAGAAATCGGTGGCCGATCA 1 M T G L Y D G I P V T E K S V A D 181 CCCGAAGGGCCGGACATTATCTGGCTGCCCACCGGTGGAGGAAAAACCCGTGCTGCGGT 18 Q P E G P D I I W L P T G G G K T R A A V 241 TTACATCACCAAGAAACACCTGGAGACCACAGCCAATGCCAAAGTAGCAGTGCTCGTCAA 38 Y I T K K H L E T T A N A K V A V L V N K 301 AAGGTGCATCTGGTAGATCAGCACTTTGCAAAAGGATTCAGGCCTTATCTTGGGAGTACAT 58 V H L V D Q H F A K G F R P Y L G S T Y K 361 CAAGATAGCAGCCATCAGTGGGGACAGTAATGAGAAAGACTTGTTTGGGTGTCTAGTCAA 78 I A A I S G D S N E K D L F G C L V K A S 421 GGCCTCAGACCTGGTCATCTGCACAGCTCAGATCTTGGAGAACGCCCTGACCAACATGGA 98 D L V I C T A Q I L E N A L T N M E E E K 481 GGAAGAAAAACACGTGGAGCTGACTGACTTTACCCTGCTGGTAATAGACGAATGTCACCA 118 H V E L T D F T L L V I D E C H H T Q K E 541 TACACAAAAGGAGAGCGTCTATAATAAGATAATGGGCCGCTATGTTGAAAAGAAAGTGAG 138 S V Y N K I M G R Y V E K K V R K E G N L 601 AAAAGAAGGGAATCTTCCTCAGATTTTGGGTCTCACAGCATCGCCTGGTGCAGGAGAAAA 158 P Q I L G L T A S P G A G E N K Q L D K A 661 TAAGCAACTAGATAAAGCTGTTGAACATGTACTGCAGATCTGTGCCAATCTGGATTCAGT 178 V E H V L Q I C A N L D S V I V S T K E 721 AATTGTGTCCACCAAGGAGTTTACTCCAGTGCTGCAGAAAGTCGTTCCCAGACCCAAAAA 198 F T P V L Q K V V P R P K K Q Y D I V E 781 GCAATATGATATTTGACACACCATTTAAACTCAGTCAAAACACTGTCACAGTTACTGTGT 218 R R T L D P F G D H L K A M M L M I H E 841 ATGATGTTGATGATTCATGAGTATATGCCGCCAACGGTGAGTCGCAGCCCGCGAGAGATG 238 Y M P P T V S R S P R E M G T Q E Y E A 901 GGCACCCAGGAGTATGAAGCTGATGTGGTGGAACTAGAAAAAGAAGGTGTAAAGAAAGAG 258 D V V E L E K E G V K K E N R L I A Q C 961 AACAGACTGATTGCTCAGTGTGCTTTGCACCTGCGGCAATACAACGATGCTCTTCTCATT 278 A L H L R Q Y N D A L L I N D A V R M V 1021AATGACGCCGTTCGCATGGTGGACGCCTTCCGGGGTCTAGACGAGTTCTACAATTCAAGG 298 D A F R G L D E F Y N S R K T K L L D G 1081 AAAACCAAATTGCTGGATGGAACAGATATCTTTCTCCAGGGACTTTTTGATGAGAACCGT 318 T D I F L Q G L F D E N R V E L K Q L A 1141 GTGGAGCTTAAACAGCTGGCATCAAATGACCGCTACGAAAACCCCAAACTGGCCCAGTTG 338 S N D R Y E N P K L A Q L Q C T L Q E E 1201CAATGCACATTGCAGGAAGAGTTTAATGATGAAAACTCTCGTGCTATCCTCTTCTCAAAG 358 F N D E N S R A I L F S K T R R G T H C 1261ACCCGCAGGGGCACCCATTGTCTGTTTGACTGGGTGAACTCCAACCCTGAGCTGCAGAGG 378 K T R R G T H C L F D W V N S N P E L Q 1321 GTCAACATCAGAGCTGGCATTCTAACAGGAGCGGGTACAGGTGCAAATCACATGACCCAG 398 R V N I R A G I L T G A G T G A N H M T

Fig. 1. Continued.

1381AATGAACAGAAGGAAACCATAAAACATTTTAGAACTGGAATCCTCAACCTCCTCATCTCC 418 Q N E Q K E T I K H F R T G I L N L L I 1441ACCAGTGTAGCTGAGGAAGGACTTGATATTCCAGAATGCAACTTAGTTGTACGTTATGGG 438 S T S V A E E G L D I P E C N L V V R Y 1501 CTGTTGACCAATGAGATCGCTCAGCAGCAGGCCAGTGGGCGGGCTCGAGCTCTGAACAGC 458 G L L T N E I A Q Q Q A S G R A R A L N 1561 GTCTACTCAGTGGTGGCCGAAGAAGGTGGGCGTGAAATACGCAGGGAACTTACCAATGAG 478 S V Y S V V A E E G G R E I R R E L T N 1621 TATCTAGAAAGTCTGACTGCAAAAGCTATTGAGCAAGTGCAGCAAATGAGTCCAAGAGAG 498 E Y L E S L T A K A I E Q V Q Q M S P R 1681 TTTAGACACAAGATATCCGAGCTCCAGCAAATCGCTGTTCTGATTCGAATCCAGGGCGAG 518 E F R H K I S E L Q Q I A V L I R I Q G 1741 AGGAAAAAGGATGAGAGGAAGCAACGCTATAGTCCTGCTCAGGCTCAGTTTCAGTGCAGA 538 E R K K D E R K Q R Y S P A Q A Q F Q C 1801 GGATGCTTCTTGCCTGTCTGCAGTGGAGAAGATTTAAGGAAAATAGAAAATACACACCAT 558 R G C F L P V C S G E D L R K I E N T H 1861 GTCAACATCAATCCTGAGTTTGTGAGGCATTACAGAACAGGTGGGCAGGTCTTTGTGGGG 578 H V N I N P E F V R H Y R T G G Q V F V 1921 AGGAACTTTGAAGATTGGGAGCCTGGACGGGTTATCAACTGCAACAAATGTGGAAAGGAC 598 G R N F E D W E P G R V I N C N K C G K 1981 TGGGGAATGGAGATTAGATACAGGAATGTGGTAATACTCCCCTGCTTAAAAATAAAAAGC 618 D W G M E I R Y R N V V I L P C L K I K 204 1 TTTTCCTTGAAGACTCCTGAAGGCAAATCCACTCCCAAGCAGTGGAAGGATGTTGAGTTC 638 S F S L K T P E G K S T P K Q W K D V E 2101 CAGGTGAAAGAGTTCGACTTTCTTGAGGACATGAGCAGTCGTTTCCCTGACCTAAACTTA 658 F Q V K E F D F L E D M S S R F P D L N 2161 AACGACTGACACACCATTTAAACTCAGTCAAAACACTGTCACAGTTACTGTGTCAGAATT 678 L N D* 2221 TCTCTGTCTTCAGTGAGTACTTGTGTAACACTGGTTTTCTACTTGTAGGTCATGGAGACT 2281 TTGCAAGTGGCAATTAATTGCTGTTAATTCCAAACTGCTTAGCATCATTTGAATATGACA 2341 TGTTCATTCTAATTATGCCGATTATCCATCGTGGTTGTGATTTGGTCCATTGTGCTTGGT 2401 TGTGAGTGTCAAAAGACATTTAGGAATCTCTGTTCCACATAATATATCTGTACTTGACAT 2461 ATAGTTTTAAATTGTAGTGTTTACTTAGTAGTTTCTGTCATGTGAGAGTTTTGTTTGCTT 2501 TTGTCTGAGAAAGGTGTGTGTGTGTGTGTGTGTGTAAGTGTTGTGATGGTATTTTTATTT 2561 TGAACAGTCTCGTTAATTATTCTGGTGTCCCTCTCTGGGATCAGGTCTGTGCTGCCCAGA 2621 TTATGAGTTATTATAGACATTACTGTGCAATGTGTCTCTAAATGTTTCTGAAGGTCTTAA 2681 TGGCCCTCGTTGAGCTCTAGTCTGCTCAGACTGTCGCTTTCAGATTGCATGAACATCTAC 2741 ATATATTCATTTAACAGATGCTTTTATCCTAAGTGACTTACAAATGAGGAGAAATATGAA 2801 TACGTTTCAAAATCTGTGAGTCTTCATTTAAAGACTGCTTACAGGCTTGTTTATAGCACT 2861 TAAGTATGTGTCTCACCAAACATAAAATTATAATGCGTATAATGCATGCCTTTTTAAAAG 2921 TCTGACCTTTTTACATTTCTGCATTACCACATGAGTCTGTATATCACATCTAGAATATAT 2981 GATTTTTCAAAAATATATAATAAAACAATAATGCACTGTAAAAAAAAAAAAAAAAAAAAA 3061 A

Fig. 1. Nucleotide and deduced amino acid sequences of Cyprinus carpio lgp2. The nucleotide and amino acid sequences are numbered. A termination codon (TAA) in front of the start codon is underlined with a dotted line. The start codon (ATG) is underlined and the stop codon (TGA) is marked (*). Four ATTTA and two TATAA motifs in the 3′ untranslated region are indicated by boxes. The polyadenylation signals AATAAA are in bold font. In the deduced amino acid sequence, the putative Mg2+-binding sites are in bold (120–124 aa). The DExDC domains are boxed (10–221 aa), the Res III domain is underlined with a wavy line (11–176 aa), the HELICc domain is underlined in bold (396–473 aa) and the regulatory domain (RD) is indicated by shading (551–672 aa).

PHYLOGENETIC AND HOMOLOGY ANALYSIS OF RLRS To study the molecular evolution and compare the sequence homologues, all the known and predicted RLR family protein sequences in GenBank were selected to con- struct a phylogenetic tree (Fig. 2) .These results show that the RLR family proteins could be divided into Rig-I, Mda5 and Lgp2 branches. Each group included several sub-groups, and the piscine sub-group was separated from other vertebrate member sub-groups in the corresponding branch. Cyprinus carpio Lgp2 had highest amino acid identity to C. idella and C. auratus Lgp2 (77⋅7%), whereas it had lowest identity with the mouse Mus musculus (46%) (Table II).

TISSUE DISTRIBUTION OF LGP2 MRNA lgp2 expression levels in tissues including muscle, spleen, gill, intestine, brain, head kidney, skin, heart and liver were quantified by q-PCR. lgp2 mRNA transcripts were detected in all examined organs, but expression levels differed among them (Fig. 3). lgp2 mRNA expression was highest in gill, followed by intestine, brain and heart, with the least expression in skin.

KOI HERPES VIRUS DETECTION KHV was detected by PCR using specific primers. Infected C. carpio produced a single band of 290 bp, as expected for KHV DNA. Comparison with the published GenBank nucleotide sequence for cyprinid herpesvirus 3 showed 98⋅9% homology.

TIME-DEPENDENT EXPRESSION OF LGP2, MAVS, AND IRF3 MRNA FOLLOWING KHV INFECTION Severe symptoms of KHV infection, including necrotic gill lesions, interstitial nephritis, splenitis and enteritis, were evident at 3 days post-infection. Total RNA was extracted from muscle, intestine, gill, head kidney, skin, liver, heart, brain and spleen of infected C. carpio. The mRNA expression patterns of lgp2, mavs and irf3 at 24, 48, 72 and 0 hpi (control) were examined by q-PCR (Fig. 4). The expression patterns of lgp2 were similar in all examined organs, with the excep- tion of the intestine [Fig. 4(a)]. lgp2 mRNA expression increased at 24 hpi in muscle, spleen, gill, brain, skin, heart, liver and head kidney, and reached maximum induction levels of 4⋅30 fold in muscle, 10⋅00 fold in spleen, 6⋅73 fold in gill, 6⋅70 fold in brain, 6⋅84 fold in skin, 5⋅60 fold in heart and 8⋅43 fold in liver at 48 hpi, and 22⋅59 fold in head kidney at 24 hpi. Levels declined at 72 hpi in all organs except intestine, but did not return to the original levels, and remained at an increased level in the intestine at 72 h. The expression levels of C. carpio mavs were similar in muscle, spleen, gill, intestine, brain, skin and head kidney [Fig. 4(b)]. Cyprinus carpio mavs mRNA expression showed initial increase at 24 hpi and had returned to the original level at 72 hpi. Cypri- nus carpio mavs mRNA reached maximum induction of 16⋅0 and 2⋅8 fold at 24 hpi in spleen and brain, respectively. Although C. carpio mavs mRNA expression levels were originally highest in liver, these levels reduced following challenge by KHV. As indicated in Fig. 4(c), constitutive expression of irf3 was ubiquitous in all the examined tissues. q-PCR showed that irf3 expression levels were similar in all tissues, but higher in the brain. irf3 mRNA was dramatically up-regulated at 24 hpi and reached

69 Homo sapiens MDA5 100 Pongo abelii MDA5 P 45 Macaca mulatta MDA5 22 Lepus granatensis MDA5 100 Sylvilagus bachmani MDA5 14 Pteropus alecto MDA5 94 Oreochromis niloticus Mda5 P Physetev catodon Mda5 P 91 75 Capra hircus MDA5 P 23 Sus scrofa MDA5 100 Trichechus manatus latirostris Mda5 P 34 Elephantulus edwardii Mda5 P Rattus norvegicus MDA5 91 100 Mus musculus transcript1 MDA5 100 Mus musculus transcript2 MDA5 100 Anser cygnoides Mda5 95 Gallus gallus Mda5 100 Alligator mississippiensis Mda5 P 77 Chelonia mydas Mda5 P 100 Chrysemys picta bellii Mda5 78 Chilo scyllium griseum Mda5 100 Carassius auratus Mda5 100 Ctenopharyngodon idella Mda5 92 Danio rerio Mda5 P Ictalurus punctatus MDA5 P 100 Takifugu rubripes Mda5 FishMda5 Callorhinchus milii Mda5 P 73 88 Paralichthys olivaceus Mda5 62 Epinephelus coioides Mda5 100 46 Oreochromis niloticus Mda5 P 93 Sus scrofa LGP2 100 Bos taurus LGP2 100 Pteropus alecto LGP2 Mus musculus LGP2 75 100 Homo sapiens LGP2 100 Rattus norvegicus LGP2 Gallus gallus Lgp2 100 Carassius auratus Lgp2 87 Ctenopharyngodon idella Lgp2 100 100 Cyprinus carpio Lgp2 67 Danio rerio Lgp2 Ictalurus punctatus Lgp2 Fish Lgp2 99 Gadus morhua Lgp2 Partial Paralichthys olivaceus Lgp2 86 79 Salmo salar Lgp2 100 Oncorhynchus mykiss Lep2 splicing variant2 partial 98 Oncorhynchus mykiss Lep2 splicing variant1 partial 100 Carassius auratus Rig-1 86 Ctenopharyngodon idella Rig-1 100 Cyprinidae sp. EPC Rig-1 Fish Rig-1 95 Cyprinus carpio Rig-1 100 Astyanax mexicanus Rig-1 P 88 Ictalurus punctatus Rig-1 Salmo salar Rig-1 29 Latimeria chalumnae Rig-1 49 Callorhinohus milii Rig-1 P Xenopus (Silurana) tropicalis Rig-1 P 100 Chrysemys picta bellii Rig-1 P 100 Chrysemys picta bellii Rig-1 100 Chelonis mydas Rig-1 P 73 99 Pelodiscus sinensis Rig-1 100 Alligator mississippiensis Rig-1 P 100 Alligator sinensis Rig-1 P Anser anser breed Yangzhou Rig-1 Omithor hynchus anatinus RIG-1 P 64 98 Mus musculus RIG-1 100 Rattus norvegicus RIG-1 84 Cricetulus griseus RIG-1 100 Lepus europaeus RIG-1 100 Sylvilagus bachmani RIG-1 84 Trichechus manatus latirostris RIG-1 83 Orycteropus afer afer RIG-1 P Erinaceus europaeus RIG-1 P 64 100 Leptonychotes weddellii RIG-1 P 26 Odobenus rosmarus divergens RIG-1 P 25 Homo sapiens RIG-1 100 Macaca mulatta RIG-1 31 Pteropus alecto RIG-1 Sus scrofa RIG-1 50 Bubalus bubalis RIG-1 66 Balaenoptera acutorostrata scammoni RIG-1 P 47 100 Lipotes vexillifer RIG-1 P 97 Orcinus orca RIG-1 P Cyprinus carpio Th3

Fig. 2. Continued.

© 2014 The Fisheries Society of the British Isles, Journal of Fish Biology 2014, doi:10.1111/jfb.12541 10 X. L. CAO ET AL. a maximum at 48 hpi in spleen, gill and brain, and at 24 hpi in head kidney (27⋅00 fold). Levels of irf3 expression returned to basal levels by 72 hpi in spleen and head kidney, and were decreased to 5⋅50 fold original levels at 72 hpi in intestine.

LGP2 MRNA EXPRESSION PROFILES IN VITRO AFTER POLY(I:C) STIMULATION lgp2 mRNA expression levels were examined in EPC cells at 0, 2, 8 and 24 h after stimulation with the dsRNA mimic poly(I:C) [Fig. 5(a)]. lgp2 mRNA expression was

Fig. 2. Phylogenetic relationships of retinoic acid-inducible gene-1-like receptor (RLR) family protein sequences in GenBank, employing common carp Tlr3 sequence as an outgroup. Multiple alignment of the full-length or partial protein sequences of known or predicted Rig-I, Mda5 and Lgp2 was generated by CLUSTALW and used for construction of a phylogenetic tree using the neighbour-joining method within the Mega 4.0.2 programme. Data were analysed using Poisson correction. The bootstrap values of the branches were obtained by testing the tree 1000 times. The bar indicated the distance. The ‘P’ after the species name indicated the sequence was predicted. The ‘partial’ behind the species name meant the protein was a partial sequence. The protein IDs were as follows: Homo sapiens Mda5 AF095844.1; Pteropus alecto Mda5 JN031515.1; Macaca mulatta Mda5 DQ875603.1; Lepus granatensis Mda5 KF640622.1; Sylvilagus bachmani Mda5 KF640621.1; Oreochromis niloticus Mda5 XM_003447407.2; Capra hircus XM_005676042.1; Trichechus manatus latirostris Mda5 XM_004375429.1; Elephantulus edwardii XM_006879359.1; Rat- tus norvegicus XM_006234286.1; Mus musculus transcript 1 NM_027835.3; Mus musculus transcript 2 NM_001164477.1; Anser cygnoides Mda5 KC282429.1; Gallus gallus MDA5 AB371640.1; Alligator mississippiensis Mda5 XM_006277996.1; Chelonia mydas Mda5 XM_007056641.1; Chrysemys picta bellii Mda5 XM_005290355.1; Chiloscyllium griseum Mda5 HG964652.1; Carassius auratus Mda5 JF970226.1; Ctenopharyngodon idella Mda5 FJ542045.2; Danio rerio Mda5 XM_689032.4; Takifugu rubripes Mda5 XM_003961960.1; Callorhinchus milii Mda5 XM_007889844.1; Ictalurus punctatus Mda5 JQ008942.1; Oncorhynchus mykiss Mda5 NM_001195179.1; Epinephelus coioides Mda5 HQ880665.1; Paralichthys olivaceus Mda5 HQ401014.1; Oreochromis niloticus XM_003447407.2; Carassius auratus (accession number: JF970227.1); Ctenopharyngodon idella Lgp2 FJ813483.2; Danio rerio Lgp2 NM-001257157.1; Ictalurus punctatus Lgp2 JQ008941.1; Paralichthys olivaceus Lgp2 HM070372.1; Salmo salar Lgp2 BT045378.1; Pteropus alecto Lgp2 JN031516.1; Oncorhynchus mykiss Lgp2 partial splicing variant 1 FN396358.1; Oncorhynchus mykiss Lgp2 partial splicing variant 2 2FN396359.1; Gallus gallus Lgp2 HQ8455773.1; Gadus morhua Lgp2 partial EU371924.1; Mus musculus NM_030150.2; Homo sapiens Lgp2 NM-024119.2; Xenopus laevis Lgp2 NP_01085915; Rattus norvegicus Lgp2 XM_006247315.1; Sus scrofa Lgp2 FJ392007.1, Bos taurus Lgp2 BC146128.1; Macaca mulatta Lgp2 XP_001108799; Anas platyrhynchos Lgp2 JQ868804.1; Ctenopharyngodon idella RIG-I ADC81089; Danio rerio Rig-I XP_002666571; Cyprinus carpio Rig-I HQ850439.1; Salmo salar Rig-I NM_001163699.1; Cyprinidae sp. EPC Rig-I FN394062.1; Chiloscyllium griseum Rig-I HG964657.1; Gadus morhua Rig-I HM046436.1; Ictalurus punctatus Rig-I JQ008940.1; Carassius auratus Rig-I JF970225.1; Anas platyrhynchos Rig-I ACA61272; Pelodiscus sinensis Rig-I XM_006123091.1; Latimeria chalumnae Rig-1 XM_006009537.1; Callorhinchus milii Rig-I XM_007909503.1; Xenopus (Silurana) tropicalis Rig-1 XM_002935671.2; Chry- semys picta bellii Rig-I XM_005294762.2; Anser anser breed Yangzhou Rig-I HQ829831.1; Latime- ria chalumnae Rig-1 XM_006009537.1; Balaenoptera acutorostrata scammoni Rig-I XM_007183182.1; Bubalus bubalis Rig-I XM_006060270.1; Pteropus alecto Rig-I JN031514.1; Trichechus manatus latirostris Rig-I XM_004390933.1; Orycteropus afer afer Rig-I XM_007950699.1; Leptonychotes weddellii Rig-I XM_006747115.1; Odobenus rosmarus divergens Rig-I XM_004392173.1; Homo sapiens Rig-I NP_055129; Macaca mulatta Rig-I NP_001036133; Sus scrofa Rig-I ABV26717; Lipotes vexillifer Rig-I XM_007472300.1; Orcinus orca Rig-I XM_004283615.1 ; Erinaceus europaeus Rig-I XM_007531244.1; Mus musculus Rig-I NP_766277; Rattus norvegicus Rig-I NP_001100115; Lepus europaeus Rig-I KF640636.1; Sylvilagus bachmani Rig-I KF640631.1; Astyanax mexicanus Rig-I XM_007237377.1; Ornithorhynchus anatinus Rig-I XM_007671324.1 and Cyprinus carpio Tlr3 DQ885910.1.

© 2014 The Fisheries Society of the British Isles, Journal of Fish Biology 2014, doi:10.1111/jfb.12541 LGP2 ANTIVIRAL RESPONSE 11 5 5 3 3 1 8 6 0 0 0 4 . ⋅ ⋅ ⋅ ⋅ ⋅ ⋅ ⋅ ⋅ ⋅ ⋅ ⋅ ID 246 275 945 044 276 246 649 049 546 347 ⋅ ⋅ ⋅ ⋅ ⋅ ⋅ ⋅ ⋅ ⋅ ⋅ ID 91 376 345 644 279 547 949 750 347 348 ⋅ ⋅ ⋅ ⋅ ⋅ ⋅ ⋅ ⋅ ⋅ SR M ID 76 49 847 146 044 647 749 448 048 7 . ⋅ ⋅ ⋅ ⋅ ⋅ ⋅ ⋅ ⋅ H ID 82 48 947 347 047 848 6 548 749 iss sapiens scrofa norvegicus musculus . ⋅ ⋅ ⋅ ⋅ ⋅ ⋅ ⋅ k O ID 45 086 360 461 061 261 461 . ⋅ ⋅ ⋅ ⋅ ⋅ ⋅ P ID 63 860 162 962 962 659 . ⋅ ⋅ ⋅ ⋅ ⋅ S ID 68 163 066 965 464 ⋅ ⋅ ⋅ ⋅ . I ID 64 565 866 665 . ⋅ ⋅ ⋅ D ID 65 775 776 . ⋅ ⋅ C ID 76 777 . ⋅ C ID 99 . C ID 77 carpio idella auratus rerio punctatus salar olivaceus my iss k Table II. Homologous comparisons of Lgp2 with other known piscine, sea urchin, human and mouse Lgp2 protein sequences (%) Sequences Cyprinus carpio ID, identity. Salmo salar Paralichthys olivaceus Ctenopharyngodon idella Oncorhynchus my Homo sapiens Sus scrofa Rattus norvegicus Carassius auratus Mus musculus Danio rerio Ictalurus punctatus

5 4·5

RNA 4 m

n 3·5 e cti a - ssu 3 b

p2- 2·5 ion ti g s l 2 e of g expan 1·5 1 0·5 Fold chan Fold 0 Muscle Spleen Gill Intestines Brain Head kidney Skin Heart Liver Tissue

Fig. 3. Tissue distribution of lgp2 mRNA in healthy Cyprinus carpio. lgp2 mRNA levels were measured by quantitative real-time polymerase chain reaction (q-PCR) and normalized against the housekeeping gene 𝛽-actin. Values are mean ± s.d. (n = 5).

rapidly and significantly elevated at 2 h⋅ (13 63-fold, P < 0⋅05), and then gradually decreased. The transcripts returned to control levels at 24 h post-stimulation (P < 0⋅05).

TEMPORARY EXPRESSION PATTERN OF LGP2INVITROAFTER KHV INFECTION Plaques were observed at 8 hpi with KHV,and then spread gradually. lgp2 expression in EPC cells was significantly enhanced at 8 ⋅h (1 77 fold, P < 0⋅05), reached a peak at 24 h (2⋅16 fold, P < 0⋅05) and returned to control levels at 48 h (P > 0⋅05) [Fig. 5(b)]. There were no significant differences in lgp2 mRNA expression levels among the different time intervals in corresponding control samples (P > 0⋅05).

DISCUSSION lgp2 is known to play an essential role in antiviral responses (Honda & Taniguchi, 2006). Cyprinus carpio lgp2 has been cloned from C. auratus, D. rerio (Sun et al., 2011), pufferfish Takifugu rubripes (Temminck & Schlegel 1850) (Zou et al., 2009) and C. idella (Huang et al., 2010). Gene cloning in this study allowed full-length lgp2 cDNA sequences to be obtained, which were predicted to encode a 680 aa protein (the- oretical pI/MW: 6⋅53/77 kDa). Six AU-rich instability motifs that might be involved in RNA instability and translational control were found in the 3′ UTR of lgp2. Stud- ies have shown that these motifs played a role in the 3′ end processing of pre-mRNA and mRNA deadenylation and decay (Beyer et al., 1997; Key & Pagano, 1997; Xu et al., 1997). In addition, four alternative splicing variants of lgp2 were obtained by gene cloning in a recent study (unpubl. data), which might explain why these motifs confer lgp2 mRNA instability, although their involvement in the antiviral response in C. carpio remains to be determined. The predicted Lgp2 protein sequence included the conserved DExDc, ResIII, HELICc and RD domains, implying similar fundamen- tal functions. The Asp-Glu-X-Asp–His (DExD-H) motif in Lgp2 is highly conserved

40 (a) f 30

20 e d d d c d d c c b c c c c c c c c c c 10 b s b b b b b e a a a a a a a a a a ssu 0

f 40 ion in ti

ss (b) e e e 30

d e e e e d e RNA expre RNA c d d d d d d d c d d d m

n c d d c cti c c a c c c - b b b b b

10 3 f

n a a i a a

e of 0 g

40 (c) g f Fold chan Fold f 30 f e f

e e 20 ee

b c d b bc b b c b c c c c 10 a a a a a b b b b b a a a a 0 0 24 48 72 0 24 48 72 0 24 48 72 0 24 48 72 0 24 48 72 0 24 48 72 0 24 48 72 0 24 48 72 0 24 48 72 Muscle Spleen Gill Intestine Brain Head kidney Skin Heart Liver Time (h) post KHV injection in examined tissues

Fig. 4. Expression of (a) lgp2,(b)mavs and (c) irf3 mRNA in muscle, intestine, gill, head kidney, skin, liver, heart, brain and spleen of koi herpesvirus (KHV)-infected Cyprinus carpio at 0, 24, 48 and 72 h post-infection. Expression levels of lgp2, mavs and irf3 mRNA were measured in tissues from KHV-infected fish by quantitative real-time polymerase chain reaction (q-PCR) at 0, 24, 48 and 72 hpi, and were normalized against the housekeeping gene 𝛽-actin. Values are mean ± s.d. (n = 5). Different lower-case letters indicate significant differences, P < 0⋅05. in fishes and mammals, with the consensus sequence Asp-Glu-Cys-His (DECH). The lgp2 gene showed high homology with lgp2 in C. idella and C. auratus (77⋅7%). The phylogenetic tree also showed that C. carpio, D. rerio, C. idella and C. auratus belong to the same cluster, which is most closely related to the cluster including I. punctatus, rainbow trout Oncorhynchus mykiss (Walbaum 1792) and S. salar, and thus formed a separate cluster of fish Lgp2 differing from other vertebrate Lgp2s. Rig-I, Mda5 and Lgp2 have recently been identified as essential for the recognition of and response to viral pathogens, establishing their importance for innate antiviral immunity (Yoneyama et al., 2005; Kato et al., 2006; Takahasi et al., 2008). The significant role of these proteins in antiviral signalling makes them prime targets for virus-encoded host invasion and antagonism. The role of Lgp2, which

(a) 20 s

a * 15 lated cell p2–ef1 g l mu

ti 10 s RNA e of

g * m

ion in 5 ss Fold chan Fold expre 0 0 2 8 24 Time (h) post poly(I:C) stimulation (b) 20 s a * 15 * p2–ef1 g l 10 RNA e of * g m ion in infected cell

ss 5 Fold chan Fold expre 0 0 2 8 16 24 48 Time (h) post KHV infected

Fig. 5. (a) lgp2 expression in epithelioma papulosum cyprini (EPC) cells stimulated by poly(I:C). lgp2 expression was measured at 0, 2, 8 and 24 h post-stimulation and the mRNA levels were normalized to elongation factor 1𝛼 (ef1𝛼) expression. The poly(I:C) concentration was 5 mg ml−1.(b)lgp2 expression in Cyprinus carpio EPC cells infected with koi herpesvirus (KHV). lgp2 mRNA levels were measured at 0, 2, 8, 16, 24 and 48 h post-stimulation and normalized to ef1a expression. *P < 0⋅05 between experimental group and control group. Values are mean ± s.d. shows high sequence similarity to Rig-I and Mda5 and selectively binds dsRNA but lacks caspase activation and recruitment domain (CARD) effector domains for mavs signalling, has been under intense investigation. When LGP2 was discovered in mammals, in vitro studies suggested that it functioned as a negative regulator (Rothenfusser et al., 2005; Yoneyama et al., 2005; Saito et al., 2007) in Rig-I signalling. In addition, LGP2−/− M. musculus displayed enhanced resistance to intranasal infection with a lethal inoculum of vesicular stomatitis virus, known to be sensed by Rig-I. In contrast to the above mechanism, Lgp2 deficiency resulted in a suppressed Ifn response against encephalomyocarditis virus, which triggered Mda5-mediated antiviral signalling (Gitlin et al., 2006; Venkataraman et al., 2007). These results suggest that Lgp2 has both negative and positive regulatory effects on Rig-I and Mda5 signalling. The generation of LGP2−/− M. musculus and M. musculus with a point mutation, D30A, that disrupts the ATPase activity of Lgp2, revealed that Lgp2 positively regulates the production of type I Ifn in response to RNA viruses recognized by both Rig-I and Mda5 (Satoh et al., 2010). Nevertheless, Lgp2 is dis- pensable for type I Ifn production following transfection by synthetic RNAs. These results suggest that Lgp2 may modify viral RNA by removing proteins from viral ribonucleoprotein complexes or unwinding complex RNA structures to facilitate Mda5-mediated and Rig-I-mediated recognition of dsRNA (Takeuchi & Akira, 2010). Pollpeter et al. (2011) surmised that Lgp2 may function upstream of Rig-I and Mda5

© 2014 The Fisheries Society of the British Isles, Journal of Fish Biology 2014, doi:10.1111/jfb.12541 LGP2 ANTIVIRAL RESPONSE 15 and its adaptor, Mavs, as a positive regulator in viral DNA-mediated responses. In bony fishes such as P. olivaceus, C. idella and O. mykiss, expression levels of the lgp2 gene and type I ifn gene were dramatically induced by poly(I:C) stimulation and viral infection (Ohtani et al., 2010; Huang et al., 2010; Chang et al., 2011; Hikima et al., 2012). There is conflicting evidence, however, for example, in C. auratus that lgp2 inhibits poly(I:C)-induced activation of D. rerio type I ifn production (Sun et al., 2011). These data suggest that lgp2 antiviral function may vary among species of teleosts. Further studies are needed to clarify the role of lgp2; however, the present data expand the role of lgp2 to DNA-pathogen detection within cells, and further corroborate the idea of a positive regulatory role for lgp2 in vivo. KHV is a dsDNA herpesvirus-like pathogen. Risk of the disease is high in popula- tions of common carp and koi. The virus may enter the body through the gills, replicate there, and induce mucosal sloughing and necrosis. To test the involvement of lgp2 in cellular innate immune responses to pathogens with DNA genomes, lgp2, mavs and irf3 mRNA levels were measured after KHV challenge. The temporary expression pattern of lgp2 in vitro after infection was observed. lgp2 was constitutively expressed in various organs, as expected given its crucial role in antiviral immunity. Although spleen, liver and head kidney are important organs in terms of innate immunity, the lgp2 gene was not highly expressed in these tissues. Transient expression of lgp2 was observed after intraperitoneal injection of KHV, infection of EPC with KHV and poly(I:C) stimulation. lgp2 transcripts were significantly elevated at 24 h(P < 0⋅05), and continued to be elevated at 48 h (P < 0⋅05), in all tissues after KHV injection, while lgp2 transcripts in EPC cells were rapidly up-regulated following poly(I:C) stimulation, subsequently recovering to control levels. lgp2 mRNA expression in EPC cells was significantly up-regulated at 8 hpi (P < 0⋅05) and returned to control levels at 48 hpi (P > 0⋅05). Accordingly, the dramatic up-regulation of lgp2 transcription could play a key positive role in the antiviral response. Consistent with cytoplasmic viral sensor lgp2 expression, the expression profiles of the adaptor molecules mavs [also known as IFN-𝛽 promoter stimulator 1 (ips-1), virus-induced signalling adaptor (visa) and CARD adaptor induc- ing interferon-𝛽 (cardifs)] and irf3 were similar in some examined tissues with respect to time-dependent induction by KHV. mavs is a CARD family protein that mediates CARD-dependent interactions with rig-I and mda5. These interactions stimulate mavs signalling of downstream irf3 and nuclear factor-𝜅B transcription factors (nf-𝜅b) that induce IFN-a/𝛽 production and IFN-stimulated genes that suppress virus infection. lgp2, however, lacks the two CARDs present at the N-termini of rig-I and mda5 which mediate homotypic interactions involved in the assembly of a complex with the essential RLR adapter Mavs and other downstream molecules. Given that mavs and irf3 expression levels were dramatically induced, it was surmised that RLR participates in sensing and signalling in response to viral DNA. Based on overall results, it was spec- ulated that lgp2 might contribute to the positive regulation of dsDNA-virus-mediated cellular innate immune responses. There are two possible mechanisms whereby lgp2 up-regulation could be involved in the antiviral innate immune response: lgp2 could work upstream of rig-I and mda5 to potentiate viral DNA-induced signalling, or lgp2 may be a node or branch point for co-ordinating crosstalk among seemingly diverse innate responses that are activated in infected cells. In summary, this study identified lgp2 as a component of innate cellular responses mediated by DNA pathogens, and demonstrated, both in vitro and in vivo, that lgp2 plays an unanticipated role in the positive regulation of downstream responses. Further

© 2014 The Fisheries Society of the British Isles, Journal of Fish Biology 2014, doi:10.1111/jfb.12541 16 X. L. CAO ET AL. investigation is needed to explore the antiviral mechanisms and regulatory pathway of lgp2 in C. carpio. Understanding these regulatory pathways might help in the development of new methods for preventing infections in C. carpio culture.

These studies were supported by the National Natural Science Foundation of China (NSFC-Henan Joint Training Fund for Fostering Talents) (number U1204329).

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