Journal of Fish Biology (2015) 86, 431–447 doi:10.1111/jfb.12559, available online at wileyonlinelibrary.com

A toll-like receptor 3 homologue that is up-regulated by poly I:C and DNA virus in turbot Scophthalmus maximus G.-B. Hu*†‡, X.-P. Li*, D.-H. Liu§, Q.-M. Liu* and S.-C. Zhang*† *College of Marine Life Sciences, Ocean University of China, Qingdao 266003, China, †Institute of Evolution & Marine Biodiversity, Ocean University of China, Qingdao 266003, Chinaand §First Institute of Oceanography, State Oceanic Administration of China, Qingdao 266061, China

(Received 25 March 2014, Accepted 21 September 2014) In this study, the gene and promoter sequences of turbot Scophthalmus maximus (Sm) toll-like receptor 3 (Tlr3) were cloned and its mRNA tissue distribution and gene expression in response to polyinosinic:polycytidylic acid (poly I:C) and turbot reddish body iridovirus (TRBIV) challenges were studied in vivo. The smtlr3 gene spans over 4⋅4 kb with a structure of five exons–four introns and encodes a peptide of 916 amino acids. The putative protein shares the highest sequence identity of 52⋅8–78⋅5% with fish Tlr3 and contains a signal peptide sequence, 13 leucine-rich repeat (LRR) motifs, a transmembrane region and a toll/interleukin-1 receptor (TIR) domain. Phylogenetic analysis grouped it with other teleost Tlr3s. A number of transcription factor binding sites were identified in the 1538 bp 5′ flanking region of smtlr3, including interferon-stimulated response element (ISRE) and those for interferon regulatory factors (IRF) and signal transducer and activator of transcriptions (STATs) smtlr3 transcripts were expressed ubiquitously with higher levels in the head kidney, heart and digestion organs. They were up-regulated by both poly I:C and TRBIV in immune and non-immune organs, but most strongly in the head kidney. Finally, the smtlr3 exhibited a two-wave induced expression during a five day time course when exposure of S. maximus to poly I:C. These findings provide insights into the role of SmTlr3 in antiviral response. © 2015 The Fisheries Society of the British Isles

Key words: antiviral response; gene expression; promoter characteristics; scophthalmid; Tlr3 signalling.

INTRODUCTION Toll-like receptors (TLR), belonging to the type I transmembrane (TM) proteins, are at the front line in the fight against invading microorganisms, and they are key mediators of innate immunity in both vertebrates and invertebrates (Gay et al., 2006). The responses of TLRs to pathogen-associated molecular patterns (PAMP) serve as a universal trigger for the immune system, the most crucial result of which is the induction of inflammation response factors and interferons (IFNs) via myeloid differentiation factor 88 (MyD88)-dependent or MyD88-independent signalling ‡Author to whom correspondence [email protected]

should

be

431 © 2015 The Fisheries Society of the British Isles

addressed.

Tel.:

+86

532

82032583;

email:

432

G .- B . H U E T A L.

pathways (Werling & Jungi, 2003). The TLR proteins are composed of two main domains, an extracellular leucine-rich repeat (LRR) domain and a cytoplasmic toll–interleukin-1 receptor (TIR) domain, connected by a linker known as TM domain. The cytoplasmic TIR domain has been shown to be involved in the signalling as well as in the localization of TLRs, while the LRR is involved in pathogen recognition (Bell et al., 2003; Sarkar et al., 2003). Up to now, at least 13 different TLRs have been identified from mammals and 17 from fish species (Rebl et al., 2010). Toll-like receptor 3 (TLR3), the first identified antiviral TLR, is an indispensable recognition receptor for host defence against viral infection (Alexopoulou et al., 2001). Each of the TLRs except TLR3 triggers activation of the MyD88-dependent signalling pathway; in contrast, TLR3 triggers a MyD88-independent signalling pathway via TIR domain-containing adapter inducing IFN-𝛽 (TRIF) (Akira & Takeda, 2004; Fan et al., 2008). TLR3 in mammals is involved in recognizing double-stranded (ds) RNA produced during replication of viruses (Sullivan et al., 2007). The homodimer of two TLR3 molecules binds single dsRNA via both the N- and C-terminal regions of their LRR domain (Liu et al., 2008). The TIR domain recruits TRIF to activate both type I IFN and pro-inflammatory responses. The TLR3-deficient mice inoculated with mouse cytomegalovirus had a 1000 fold augmentation of viral load in the spleen, demonstrating that TLR3 participates in the generation of protective immunity against viral infections (Tabeta et al., 2004). To date, a number of Tlr3 orthologues have been identified from teleosts including fugu Takifugu rubripes (Temminck & Schlegel 1850) (Oshiumi et al., 2003), zebrafish Danio rerio (Hamilton 1802) (Meijer et al., 2004), channel catfish Ictalurus punctatus (Rafinesque 1818) (Bilodeau & Waldbieser, 2005), rainbow trout Oncorhynchus mykiss (Walbaum 1792) (Rodriguez et al., 2005), grass carp Ctenopharyngodon idella (Valenciennes in Cuvier & Valenciennes 1844) (Su et al., 2009), common carp Cyprinus carpio L. 1758 (Yang & Su, 2010), large yellow croaker Larimichthys crocea (Richardson 1846) (Huang et al., 2011) and Japanese flounder Paralichthys olivaceus (Temminck & Schlegel 1846) (Hwang et al., 2012). In fishes, tlr3 expression is up-regulated after treatment with dsRNA analogue polyinosinic:polycytidylic acid (poly I:C), RNA viruses and gram-negative bacteria. The functional analyses of teleost Tlr3 have been carried out in D. rerio, T. rubripes and P. olivaceus, demonstrating that poly I:C stimulation causes Tlr3 to activate IFN and nuclear factor kappa B (NF-𝜅B) pathway (Phelan et al., 2005; Matsuo et al., 2008; Hwang et al., 2012), suggesting a conserved function of Tlr3 between teleosts and mammals. The information about Tlr3 in turbot Scophthalmus maximus (L. 1758) (Smtlr3), however, is scarce. Scophthalmus maximus is one of the main commercially important farmed marine fish species in northern China. Turbot reddish body iridovirus (TRBIV), a dsDNA virus, has caused tremendous losses to Chinese S. maximus farming. Because the Tlr3 signalling plays a crucial role in host’s immune responses against viral infections, the study of smtlr3 may contribute to develop strategies for the control of viral diseases of this economically important species. Here, its cloning, mRNA tissue distribution and in vivo immune responsive expression are reported. The immune challenge experiments were performed with poly I:C and TRBIV.

© 2015 The Fisheries Society of the British Isles, Journal of Fish Biology 2015, 86, 431–447

A TL R3 H O M O L O G U E I N S C O P H T H A L M U S M A X I M U S

433

MATERIALS AND METHODS F I S H A N D I M M U N E C H A L L E N G E S O F T R B I V A N D P O LY I : C Scophthalmus maximus juveniles (mean ± s.d. = 68⋅4 ± 4⋅5 g, n = 170), purchased from a local fish farm, were maintained in aerated seawater tanks at 16∘ C for 1 week before use. Poly I:C was purchased from Sigma (www.sigmaaldrich.com). TRBIV was isolated from cultured S. maximus with TRBIV disease as described by Shi et al. (2004). The viral titres were measured by a 50% tissue culture infective dose (TCID50 ) assay according to the method of Reed & Muench (1938). Two groups of S. maximus were injected intraperitoneally with poly I:C (10 mg ml−1 , 100 μl per fish) and TRBIV (2 × 106 TCID50 ml−1 , 120 μl per fish). Control fish were injected with the same volume (100 or 120 μl per fish) of phosphate-buffered saline (PBS). The intact healthy fish were used for tissue distribution analysis of smtlr3 mRNA, while the head kidney, spleen, gills and muscle of injected fish were collected at various time points post-injection (0, 3, 6 and 12 h and 1, 2, 3, 4 and 5 days after poly I:C injection or 0, 3 and 6 h and 1, 2, 3, 4 and 5 days after TRBIV injection) for gene expression assay in response to immune challenge. I S O L AT I O N O F R N A A N D G E N O M I C D N A Total RNA was extracted from various tissues of S. maximus including brain, gills, stomach, intestine, head kidney, kidney, spleen, liver, heart, gonad, muscle and skin using Isogen reagent (Nippon Gene; http://nippongene.com/) according to the instruction. RNA samples were treated with DNase I to remove genomic DNA contamination using the Turbo DNA-free kit (Ambion; www.lifetechnologies.com). The RNA concentration was determined by measuring absorbance at 260 nm, and its quality was monitored by A260 nm:A280 nm ratios > 1⋅8. The genomic DNA was isolated from the muscle by a standard phenol–chloroform extraction method (Palumbi et al., 1991). C L O N I N G O F G E N E A N D P R O M O T E R S E Q U E N C E S O F S M T L R3 From 1⋅0 μg of total RNA extracted from the head kidney of a S. maximus, a ds cDNA pool was produced using a Switching Mechanism At 5′ end of RNA Transcript (SMART) cDNA Library Construction Kit (Clontech; www.clontech.com). On the basis of the conserved sequences of known fish Tlr3s, degenerate primers were designed. A 1898 bp core cDNA sequence of smtlr3 was obtained by homology cloning and a 3′ end fragment by a rapid amplification of cDNA end (RACE) method. Subsequently, two partial genomic sequences were obtained by a routine polymerase chain reaction (PCR) procedure and a third genomic fragment of 3008 bp covering the 5′ end and 5′ flanking region of the gene was obtained with a Genome Walking Kit (TaKaRa; www.takara-bio.com) using the genomic DNA template. The transcription start site (TSS) was predicted by the Neural Network Promoter Prediction programme (www.fruitfly.org/seq_tools/promoter.html). The cDNA sequence harboured in the third genomic fragment was identified by a PCR amplification for the cDNA pool. The full-length cDNA and gene sequences were then compiled. The exon–intron structure was determined by alignment of the cDNA to the genomic sequence using Genetyx 7.0 software (www.genetyx.co.jp). Primers used in this group are listed in Table I. S E Q U E N C E A N A LY S I S The sequence result of smtlr3 was compared with the GenBank/EMBL database by using the basic local-alignment search tool x (BLASTx) and BLASTp search programmes (http://blast.genome.ad.Jp/). The nucleotide sequence was translated into protein sequence using DNAMAN software. The protein domains were predicted by Simple Modular Architecture Reach Tool (SMART) (http://smart.embl-heidelberg.de/). The multiple alignment of protein sequences was produced by a Clustal W programme (www.ddbj.nig.ac.jp/E-mail/ clustalw-e.html). The phylogenetic tree was depicted on the overall amino acid sequences using

© 2015 The Fisheries Society of the British Isles, Journal of Fish Biology 2015, 86, 431–447

434

G .- B . H U E T A L.

Table I. Primers used for gene cloning and expression analysis of smtlr3 Primers

Sequence (5′ → 3′ )

Tlr3-hF1

CARACGYTNAAYGTNCA RCAYAA* TCCGTGACNACRAANAR DATYTT* AARYTRCAYGGNGA RCCNTT* GCRTGDATDATRTANGCR TCRTA* GAGCATCCTGTGGTTT GTGACGTGG GGGACCAGTATATTTGCA ACACGCCAC CAGAGACACGGCCGAG AGATTGAGG CAGAGCGGTGAAGTC ATTGAATGCC GTTTGTTCATGGAGACG TCAAGGG ATCCAGATCCACTCCC CGAC GCAGGTTTGGAGGGAT CGCT AGCCCTTCTCCGCCCT AAA CCGGAGAGACAGGGC ATCAAT CCTTGGAAAACGAGAAC TGCAG CAGGAAGACCAGAACCA CAGAG CACTGTGCCCATCTACGAG CCATCTCCTGCTCGAAGTC

Tlr3-hR1 Tlr3-hF2 Tlr3-hR2 Tlr3-3′ F1 Tlr3-3′ F2 Tlr3-SP1 Tlr3-SP2 Tlr3-SP3 Tlr3-gF1 Tlr3-gR1 Tlr3-gF2 Tlr3-gR2 Tlr3-gF3 Tlr3-gR3 𝛽-actin-F 𝛽-actin-R

Target gene tlr3

Usage First round homology PCR

Nested homology PCR First round 3′ RACE PCR Nested 3′ RACE PCR Genome-walking PCR

First intron cloning

Second and third intron cloning

Fourth intron cloning

𝛽-actin

qPCR

qPCR, quantitative real-time PCR; RACE, rapid amplification of cDNA end. *N represents all four nucleotides; D, A, T or G; R, G or A; Y, C or T.

the neighbour-joining (NJ) algorithm with 1000 bootstrapping tests within MEGA version 5.0. The transcription factor binding sites in the 5′ flanking region were predicted by the TFSEARCH ver1.3 (www.cbrc.jp/research/db/TFSEARCH.html) and the AliBaba ver2.1 programmes (www.gene-regulation.com/pub/programs/alibaba2/index.html) with default parameters.

Q U A N T I TAT I V E R E A L- T I M E P C R Quantitative real-time PCR (qPCR) assay was employed to study smtlr3 mRNA tissue distribution and gene expression upon challenge with poly I:C or TRBIV in the immune-relevant and irrelevant organs. A 1⋅0 μg of total RNA from each tissue (five individuals for each time point) was reverse transcribed into cDNA by random primers using Superscript First Strand Synthesis System (Invitrogen; www.lifetechnologies.com). qPCR was performed on an ABI Prism

© 2015 The Fisheries Society of the British Isles, Journal of Fish Biology 2015, 86, 431–447

A TL R3 H O M O L O G U E I N S C O P H T H A L M U S M A X I M U S

435

7900HT Sequence Detection System (Applied Biosystems; www.appliedbiosystems.com). The PCR mixture in 20 μl volume included 1× SYBR Green Real-time PCR Mast Mix (Toyobo; www.toyobo-global.com), 0⋅2 𝜇M each of specific forward and reverse primers (Table I) and 1⋅0 μl of diluted cDNA (50 ng μl−1 ). The PCR conditions were 94∘ C for 4 min, followed by 45 cycles at 94∘ C for 40 s, 62∘ C for 30 s and 72∘ C for 25 s. For an internal control, S. maximus 𝛽-actin transcript (accession number EU686692) was amplified with primers 𝛽-actin-F and 𝛽-actin-R (Table I). All samples were amplified in triplicates. Fluorescent detection was performed after each extension step. A dissociation protocol was performed after thermocycling to verify that a single amplicon of expected size was amplified. Levels of smtlr3 were expressed as relative value to the 𝛽-actin level in the corresponding sample in the tissue distribution analysis or as fold change relative to the expression level in control according to the 2−ΔΔCT method after being normalized to the 𝛽-actin in the gene expression assay (Livak & Schmittgen, 2001).

S TAT I S T I C A L A N A LY S I S Statistical analysis was performed using SPSS13.0 software (SPSS Inc.; www.ibm.com). Differences in the data were compared by one-way analysis of variance (ANOVA) followed by Duncan’s post hoc test for multiple comparisons. Differences were considered significant at P < 0⋅05.

RESULTS M O L E C U L A R C L O N I N G A N D C H A R A C T E R I Z AT I O N O F SM T L R3

The full-length smtlr3 cDNA (GenBank accession number KJ194173) is 3482 bp long and contains an open reading frame (ORF) of 2751 bp which encodes a polypeptide of 916 amino acid residues, a 5′ terminal untranslated region (UTR) of 244 bp and a 3′ UTR of 487 bp. The 3′ UTR possesses a putative polyadenylation consensus signal (AATAAA) 14 bp upstream of the poly (A) tail and no putative ATTTA instability sequences was found in the 3′ UTR. SMART programme analysis of Smtlr3 protein structure indicated a potential signal peptide in the first 21 amino acids, followed with 13 LRR motifs at positions 56–708 in the N-terminal LRR domain including a LRR-TYP (typical LRR) and a LRR-CT (C-terminal LRR), a TM domain at positions 714–736 and an intracellular C-terminal TIR region at positions 767–911 (Fig. 1). The numbers of Tlr3 LRRs are different with different fish species ranging from 13 to 21, while the number in humans Homo sapiens is 18 (Table II). The Smtlr3 protein shares a high sequence identity of 52⋅8–78⋅5% to other teleost Tlr3s and a lower identity of 43⋅7% to human TLR3 (Table II). Similar results were obtained for the conserved TIR domain but the identities are much higher. P H Y L O G E N E T I C A N A LY S I S O F TL R3

To further study the evolutionary relations among vertebrate Tlr3s, a phylogenetic tree was conducted based on Clustal W full-length amino acid sequence alignment using the NJ method (Fig. 2 and Table III). The result indicated that Smtlr3 was in the same sub-group with the Tlr3s from other teleosts. It exhibited the closest phylogenetic distance to the sequences from P. olivaceus and L. crocea. Tlr3s from amphibians, birds and mammals were also clustered to their corresponding sub-groups. Thus, the relationships within the tree reflect well the taxonomic locations of the species.

© 2015 The Fisheries Society of the British Isles, Journal of Fish Biology 2015, 86, 431–447

436

G .- B . H U E T A L.

Fig. 1. Alignment of Scophthalmus maximus Tlr3 amino-acid sequence with those of Tlr3s from other teleosts, Paralichthys olivaceus, Larimichthys crocea, Ctenopharyngodon idella and Danio rerio, and Homo sapiens. The signal peptide, leucine-rich repeat (LRR) domains, transmembrane (TM) domain and toll–interleukin-1 receptor (TIR) domain are shown by overbars. Amino acid residues important for ligand binding are shaded, for signalling marked by , and one important for intracellular localization is marked by #. The identical residues (*), conserved substitutions (:) and semi-conserved substitutions (.) identified by the Clustal W programme are indicated. The GenBank accession numbers are shown in Table III.

© 2015 The Fisheries Society of the British Isles, Journal of Fish Biology 2015, 86, 431–447

437

A TL R3 H O M O L O G U E I N S C O P H T H A L M U S M A X I M U S

Table II. Comparison of Scophthalmus maximus Tlr3 amino-acid sequence with those of other teleosts, Paralichthys olivaceus, Larimichthys crocea, Ctenopharyngodon idella and Danio rerio, and Homo sapiens S. P. maximus olivaceus Amino-acid residues Identity (similarity)* of Tlr3 Identity (similarity)* of TIR domain Number of LRR

L. crocea

C. idella

D. rerio

H. sapiens

916 –

911 909 904 903 904 78⋅5 (87⋅8) 74⋅2 (83⋅8) 55⋅8 (72⋅5) 52⋅8 (70⋅8) 43⋅7 (60⋅8)



84⋅1 (93⋅1) 78⋅1 (88⋅4) 63⋅4 (80⋅7) 60⋅0 (78⋅6) 50⋅3 (68⋅7)

13

21

16

14

16

18

LRR, leucine-rich repeat; TIR, toll–interleukin-1 receptor; Tlr3, toll-like receptor 3. *Sequence identity and similarity are identified by EMBOSS Pair-wise Alignment Tools for Global Sequence Alignment EBI.

G E N E A N D P RO M OT E R S T RU C T U R E S O F

S M T L R3

The length of smtlr3 gene is 4438 bp (accession number KJ194174). Comparison of the cDNA and genomic sequences revealed that it has five exons and four introns. All the 5′ and 3′ ends of the introns show a canonical splicing motif (GT/introns/AG) (Breathnach & Chambon, 1981). As tlr3 in other species, the first intron of Smtlr3 is located in the 5′ UTR, while other three are in the region of ORF (Fig. 3). Although the overall structure of tlr3 is not uniform throughout vertebrates, the genomic structure for ORF part is extremely conservative, composed of four exons and three introns with the size of each exon being comparable. A 1538 bp 5′ flanking region (from −1538 to −1) was simultaneously obtained in the process of cloning 5′ terminal fragment of smtlr3 by genome walking. The predicted TSS is located at 894 bp upstream of start codon ATG, a TATA box at −27, a non-canonical CCAAT box at −68 and two GC-rich regions containing SP1 (GC box–specificity protein-1)-binding site from −385 to −360 and from −248 to −115, respectively. The IFN-stimulated response element (ISRE) and binding sites for IFN regulatory factors (IRF), signal transducer and activator of transcriptions (STATs), p53, NF-𝜅B, activator protein-1 (AP1) and PU.1/Ets (E26 transformation-specific) identified in mammalian TLR3 promoters were also found (Fig. 4). TISSUE DISTRIBUTION OF

S M T L R3 MR N A

In order to examine the tissue distribution of tlr3 mRNA in healthy S. maximus, qPCR was performed using total RNA extracted from 12 tissue types including brain, gills, stomach, intestine, heart, head kidney, kidney, liver, spleen, gonad, muscle and skin of healthy S. maximus. The results showed that smtlr3 transcripts were constitutively expressed in all the examined tissues. The highest level was observed in the head kidney, followed by the intestine, stomach and heart. Low levels were observed in the brain, spleen, gonad, muscle and skin. In the other tissues, a moderate expression was seen (Fig. 5).

© 2015 The Fisheries Society of the British Isles, Journal of Fish Biology 2015, 86, 431–447

438

G .- B . H U E T A L.

99

Ovis aries Bos taurus 37 Sus scrofa Canis lupus familiaris 57 Macaca mulatta 29 Homo sapiens 98 72 Equus caballus 100 Mus musculus Loxodonta africana Taeniopygia guttata Gallus gallus 100 Xenopus laevis 56 Paralichthys olivaceus 50 Larimichthys crocea 96 Scophthalmus maximus 92 Takifugu rubripes Oncorhynchus mykiss Ictalurus punctatus Danio rerio 67 Megalobrama amblycephala 100 Cyprinus carpio 75 Ctenopharyngodon idella 51 99 Carassius auratus 85

89

100

0·05

Mammals

Birds Amphibian

Teleosts

Fig. 2. Phylogenetic tree showing relationships of vertebrate Tlr3 proteins. The trees were constructed by the neighbour-joining method in MEGA 5.0 based on Poisson correction model with 1000 bootstrap replicates. The numbers at the branches indicate bootstrap values. The bar (0⋅05) indicates the genetic distance. The GenBank accession numbers are listed in Table III.

GENE EXPRESSION OF CHALLENGES

S M T L R3

U P O N P O LY I : C A N D T R B I V

Upon challenge with poly I:C, smtlr3 expression was down-regulated at 3 h post-injection in the gills, head kidney and muscle, but up-regulated from 6 h. The up-regulation started at 3 h in the spleen. The up-regulation continued until the end of the experiment, embracing two waves of induced expression, in all four organs. The first expression peak of smtlr3 arose at day 1 in the gills and muscle with an approximate 3⋅7 and 8⋅7 fold increase, respectively, and at 12 h in the head kidney and spleen with an approximate 8⋅4 and 3⋅2 fold increase, respectively, while the second peak arose at day 5 in all four organs with an approximate 4⋅4, 3⋅6, 10 and 3⋅3 fold increase, respectively [Fig. 6(a)–(d)]. Challenge of animals with TRBIV caused a later and a weaker induction of smtlr3, with a single expression peak observed during the five-day time course that arose very suddenly. Similar to poly I:C challenge, the smtlr3 expression was temporarily down-regulated at the beginning of the experiment, i.e. by 6 h in most organs. The expression peak was detected at days 3, 2, 1 and 1 in the gills, head kidney, spleen and muscle with an approximate 3⋅3, 4⋅3, 2⋅1 and 4⋅6 fold increase, respectively. After peak time, the smtlr3 expression gradually decreased, but at a level still over control on end date in all four organs [Fig. 6(e)–(h)].

© 2015 The Fisheries Society of the British Isles, Journal of Fish Biology 2015, 86, 431–447

A TL R3 H O M O L O G U E I N S C O P H T H A L M U S M A X I M U S

439

Table III. GenBank accession numbers of the toll-like receptor 3 (Tlr3) protein sequences used in this study Species

Accession number

Bos taurus (cattle)

ABN71659

Canis lupus familiaris (dog)

XP_540020

Species Macaca mulatta (rhesus monkey) Megalobrama amblycephala (Wuchang bream) Mus musculus (mouse) Oncorhynchus mykiss (rainbow trout) Ovis aries (sheep)

Carassius auratus (goldfish) ABC86865 Ctenopharyngodon idella ABI64155 (grass carp) Cyprinus carpio (common ABL11473 carp) Danio rerio (zebrafish) NP_001013287 Paralichthys olivaceus (Japanese flounder) Equus caballus (horse) NP_001075267 Scophthalmus maximus (turbot) Gallus gallus (chicken) ABL74502 Sus scrofa (pig) Homo sapiens (human) NP_003256 Taeniopygia guttata (zebra finch) Ictalurus punctatus ABD93872 Takifugu rubripes (fugu) (channel catfish) Larimichthys crocea (large ADZ52858 Xenopus laevis (African yellow croaker) clawed frog) Loxodonta africana ABC95781 (African savanna elephant)

Accession number ABY64988 ABI83673

AAH99937 AAX68425 NP_001129400 BAM11216 KJ194173 BAG12312 XP_002190888 AAW69373 BAF57488

DISCUSSION In this study, the cDNA and genomic sequences of a Tlr3 orthologue were cloned from the head kidney of S. maximus. Based on the results of homology search and phylogenetic analysis, the putative peptide was identified as Smtlr3. The smtlr3 cDNA consists of 3482 bp, which contains a 2751 bp ORF encoding 916 amino acids. Unlike other fish tlr3 mRNAs, no RNA instability motif (ATTTA) was found in the 3′ UTR of smtlr3, suggesting that smtlr3 mRNA may be not transiently expressed. It was reported that one, three and eight ATTTA motifs were identified in the 3′ UTR of tlr3 from D. rerio, P. olivaceus and O. mykiss, respectively. This finding indicates that the stability of tlr3 mRNA may be dissimilar in different species. The LRR domain of Smtlr3 harbours 13 LRR motifs, including 11 conserved LRRs, a LRR-TYP and a LRR-CT as analysed with the SMART programme (Fig. 1). LRRs provide a structural framework for PAMP recognition (Bell et al., 2003), while the LRR-CT module stabilizes the protein structure by protecting its hydrophobic core from exposure to solvent (Wei et al., 2011). The numbers of Tlr3 LRRs range from 13 to 21 among various species (Table II). The discrepancy in LRR number may be explained in part by the different programmes used to identify the repeats in addition

© 2015 The Fisheries Society of the British Isles, Journal of Fish Biology 2015, 86, 431–447

440

(a)

G .- B . H U E T A L.

1

100

200 LRR LRR LRR

LRR LRR

LRR

LRR

LRR TYP LRR

LRR LRR

LRR

LRR CT

TIR

(b) S. maximus 4438 bp

12/459 195

232 650

456

P. olivaceus 3085 bp

D. rerio 5284 bp

202

12/456 761

H. sapiens 15944 bp

235/463 99

1856

0·1 kb

232

0·1 kb 206

O.mykiss 7376 bp

83 148 192 83

L. crocea 5721 bp

C.idella 5668 bp

1862

1087 40/441

938 150 593

470

353 7/441

7364

1847

667 197

299 139 192 1779

3288

>0·3 kb

232/576 196

1847

502 195

26/460

94

112

153 195

431 41/442

131

154 195

229/207 252 229/754

1881 257 1852

223/1785 847

998 1853

66

229/225 472

Fig. 3. Structural features of tlr3 genes. (a) Schematic representation of Smtlr3 domains predicted by the simple modular architecture reach tool (SMART) programme. The 13 leucine-rich repeats (LRR) were located in the N-terminal region, followed by a putative transmembrane (TM) domain and a cytoplasmic toll–interleukin-1 receptor (TIR) domain at the C-terminal end. (b) Diagrammatic comparison of tlr3 genes from Scophthalmus maximus (accession number KJ194174), Paralichthys olivaceus (AB675414), Larimichthys crocea (HQ589262), Danio rerio (NC_007112), Ctenopharyngodon idella (Su et al., 2009), Oncorhynchus mykiss (AY883999) and Homo sapiens (NG_007278). Exons are represented by boxes; shady areas represent the coding region. Exon lengths in base pairs are shown on the top of each element and intron lengths on the bottom.

to the species specificity. Nevertheless, further research is needed to see whether the difference in LRR number affects the Tlr specificity for PAMP and the innate immune responses. Human TLR3 binds dsRNA at two sites of LRR domain. One site is located in the N-terminal histidine-rich region of the LRR domain, while the other nearly upstream to the C-terminal of the LRR domain (Bell et al., 2005, 2006). Three histidine residues His39 , His60 and His109 at the first dsRNA-binding site and two residues His539 and Asn541 at the second are important for ligand binding in human TLR3. As expected, these residues are highly conserved among the Tlr3 orthologues and correspond to His46 , His67 , His115 , His549 and Asn551 of Smtlr3 (Fig. 1). A motif near the C-terminal of the LRR domain (positions 555–563) provides a concave surface for dsRNA binding in human TLR3 is also conserved in Smtlr3. These suggest that the LRR domain of Smtlr3 is functional and capable of responding to mammalian TLR3 agonist dsRNA. The TIR domain is responsible for the adapter binding and signal transduction upon ligand stimulation, where Smtlr3 shares a higher identity with other Tlr3s (Table II).

© 2015 The Fisheries Society of the British Isles, Journal of Fish Biology 2015, 86, 431–447

A TL R3 H O M O L O G U E I N S C O P H T H A L M U S M A X I M U S

441

Fig. 4. The 5′ flanking region of Scophthalmus maximus tlr3 gene. The putative transcription factor binding sites are shown with bars ( ) and CCAAT and TATA boxes with boxes ( ). The putative transcription start site is indicated by a bent arrow ( ).

The Tyr759 residue was identified to be the most critical residue for the intracellular signalling in human TLR3 (Sarkar et al., 2004), which is conserved throughout all Tlr3 sequences (Tyr771 in Smtlr3) (Fig. 1). Either tyrosine residue Tyr733 or Tyr858 was important for maximal activities of the promoters of 561 genes in humans. Both are conserved in Smtlr3 (Tyr744 and Tyr868 ). Additional tyrosine residue Tyr756 reported to complement the Tyr759 function with less efficiency is also conserved in Smtlr3 (Tyr768 ). In the linker region of human TLR3 (between the TM and TIR domains with a length of c. 40 amino acids), three amino-acid residues Phe732 , Leu742 and Gly743 are known to be crucial for ligand-induced NF-𝜅B and IFN-𝛽 promoter activation (Funami

© 2015 The Fisheries Society of the British Isles, Journal of Fish Biology 2015, 86, 431–447

442 0·00035 0·0003 0·00025 0·0002 0·00015 0·0001 0·00005 0

a ab b c

ab

ab b

b c

c

c

c

Br

ai n G ill St s om ac In h te sti ne H H ea ea r d ki t dn e K y id ne y Li ve r Sp le en G on a M d us cl e Sk in

Ralative expression

G .- B . H U E T A L.

Fig. 5. Quantitative real-time (qPCR) analysis of smtlr3 transcripts in different tissues of healthy Scophthalmus maximus. The values are expressed as relative value to the 𝛽-actin levels. Values are means ± s.e. (n = 5). Values marked by different lower-case letters are significantly different from each other (P < 0⋅05).

et al., 2004), which are all found in Smtlr3 (Phe743 , Leu753 and Gly754 ) (Fig. 1). Further, the residue Arg740 in the linker region of human TLR3, identified to be important for the intracellular localization (Funami et al., 2004), is conservative throughout vertebrate Tlr3s (Arg751 in Smtlr3) (Fig. 1). Altogether, these conservation features indicate that the cytoplasmatic region of Smtlr3 may be essential for its localization and signalling. The genomic organization of smtlr3 consists of five exons and four introns, same with that of tlr3 from most species (Fig. 3). Oncorhynchus mykiss tlr3 is exceptional because its 3′ UTR is interrupted with an additional intron resulting in a six exon–five intron structure, and, apparently, P. olivaceus tlr3 is incomplete with the 5′ and 3′ UTRs unavailable. In particular, in the ORF part, the genomic organization is extremely conserved throughout vertebrates, all composed of four exons and three introns with each exon having a similar size. This finding suggests that the function of vertebrate tlr3 may be evolutionarily conserved. The smtlr3 promoter contains ISRE and STAT-binding site (Fig. 4), two cis-acting elements necessary for the basal promoter activity of TLR3 in humans and mice (Heinz et al., 2003), suggesting a possible similar role for them in smtlr3 transcriptional regulation. The NF-𝜅B-binding site was identified in murine TLR3 promoter but not in human promoter and, thus, considered to be possibly responsible for the species-specific LPS-responsiveness in murine cells (Rehli, 2002). Interestingly, the NF-𝜅B-binding site is present in promoters of S. maximus and other known teleost tlr3 genes (Rodriguez et al., 2005; Su et al., 2009), indicating that teleost tlr3 is not so divergent in transcriptional modulation as mammalian TLR3 at this point. Other elements (the binding sites for p53, PU.1/Ets and IRF1) reported for mammalian TLR3 promoters were also identified in this study. Taken together, the promoter structural features exhibited by smtlr3 suggest that the expression of fish tlr3 as well as the involved parts of immune systems might be controlled by a mechanism similar to that used by mammals. The smtlr3 transcripts were expressed ubiquitously in all examined tissues of healthy fish, with higher levels observed in the head kidney, heart and digestive organs (Fig. 5). This tissue expression profile is similar to that of tlr3 from most of the investigated teleosts including D. rerio (Phelan et al., 2005), O. mykiss (Rodriguez et al., 2005), I. punctatus (Baoprasertkul et al., 2006), rare minnow Gobiocypris rarus Ye & Fu 1983

© 2015 The Fisheries Society of the British Isles, Journal of Fish Biology 2015, 86, 431–447

443

** *

*

*

**

** *

*

*

*

*

** * 5 days

* 3 days

2 days

1 days

* 12 h

6h

3h

*

*

10 8 6 4 2 0

(f)

5 4 3 2 1 0

(g)

10 8 6 4 2 0

(h)

** *

*

** *

*

*

*

**

5 days

(d)

**

**

*

3 days

15 12 9 6 3 0

*

2 days

(c)

*

1 days

5 4 3 2 1 0

**

**

**

12 h

(b)

*

(e)

6h

15 12 9 6 3 0

*

**

5 4 3 2 1 0

3h

(a)

0h

10 8 6 4 2 0

0h

Fold induction

A TL R3 H O M O L O G U E I N S C O P H T H A L M U S M A X I M U S

Time post-injection Fig. 6. Time-course expression profiles of smtlr3, shown as fold changes, in (a, e) gills, (b, f) head kidney, (c, g) spleen and (d, h) muscle of Scophthalmus maximus challenged with (a–d) 1⋅0 mg poly I:C per fish or (e–h) 2⋅4 × 105 tissue culture infective dose (TCID50) of turbot reddish body iridovirus (TRBIV). Each data point is expressed as the mean of replicates ± s.e. (n = 5). The level of significance of the comparison to the control is indicated by *P < 0⋅05 and **P < 0⋅01.

(Su et al., 2008), C. carpio (Yang & Su, 2010) and L. crocea (Huang et al., 2011), and most similar to another flatfish P. olivaceus that also has higher tlr3 transcript levels in the head kidney and heart (Hwang et al., 2012). On the contrary, the liver was reported to express tlr3 mRNA abundantly in some teleosts (Baoprasertkul et al., 2006; Huang et al., 2011), whereas it expressed them much less in S. maximus as demonstrated in G. rarus and P. olivaceus. Further, the expression of T. rubripes tlr3 was limited to the liver and digestive organs (Oshiumi et al., 2003). Thus, the tissue expression profile of teleost tlr3 is variable. It is noteworthy that tlr3 expression was weak in the spleen in S. maximus. It is known that the spleen in teleosts is rich in erythrocytes and granulocytes, but relatively deficient in monocytes, a cell type equivalent to the progenitor of TLR3-synthetic dendritic cells and macrophages in mammals. In contrast, the head kidney contains abundant monocytes, so it is not surprising that smtlr3 expression was detected most strongly in it. In order to explore the functions of Smtlr3 in antiviral immune response, its gene expression in response to poly I:C and TRBIV challenges were investigated over five

© 2015 The Fisheries Society of the British Isles, Journal of Fish Biology 2015, 86, 431–447

444

G .- B . H U E T A L.

days in the gills, head kidney, spleen and muscle that represent both immune and non-immune organs (Fig. 6). The results showed a significant enhancement of smtlr3 mRNA level with both poly I:C and TRBIV (a virus with a linear dsDNA genome) treatments in all four organs, suggesting that both RNA and DNA viruses can transcriptionally modulate tlr3 expression and a role of smtlr3 in both immune and non-immune tissues. As far as is known, this study is the first report for induction of tlr3 by DNA virus in teleosts although one study reported its up-regulation with CpG ODN, a synthetic mimic of bacterial and viral DNA, in P. olivaceus (Hwang et al., 2012). The strongest induction was detected in the head kidney in both challenge cases, perhaps due to a large number of Tlr3-synthetic cells existing in this organ as implied by the high basal level observed in the tissue distribution analysis. The induction by poly I:C initiated apparently more quickly and was stronger in the four tested organs, suggesting that it acts as a highly pure and concentrated PAMP to activate host’s immune response directly. In contrast, TRBIV has to go through replication cycles to produce PAMPs before triggering a detectable response. Consistently, the sudden peak in TRBIV-induced smtlr3 expression may reflect a property of viral single burst curve. A temporary down-regulation of smtlr3 was found in early phase of challenges. The early temporary down-regulation was also reported for other teleost tlr3 genes after poly I:C or virus exposure (Phelan et al., 2005; Rodriguez et al., 2005; Huang et al., 2011), which could be caused by the transmission of antigen-presenting cells (APC), among other leucocytes, to the intraperitoneum in the intraperitoneal injection or an immune depression at beginning of challenge (Rodriguez et al., 2005; Yang & Su, 2010). smtlr3 had two waves of enhanced expression with poly I:C treatment, a phenomenon not described for other teleost tlr3 genes before. The reason for this may lie in that their time-course expression with poly I:C stimulation was not studied beyond 48 h when the second wave just begins. The first expression wave started early (at 3–6 h), probably due to the binding of the activated IRFs to the IRF-E/ISRE elements in the smtlr3 promoter. This process does not require new protein synthesis and is accomplished just through phosphorylation of the existing IRFs mediated by Tlr3 signalling upon recognition of poly I:C. Also, the activated IRFs can bind to the promoters of type I IFNs and IFN-stimulated genes (ISG) and lead to their transcriptions in the same process. A similar case should exist for TRBIV infection, but needs a pre-step to produce PAMPs and phosphorylates IRFs through different signal pathways, e.g. the Tlr9 and Tlr3 pathway triggered by viral unmethylated CpG-DNA and dsRNA intermediates, respectively. So, in this context, the induction of tlr3 was independent of viral genomic nucleic acid type, but showed different kinetics with different viral infection. In P. olivaceus, intracellular poly I:C stimulation causes Tlr3 to activate both IFN and NF-𝜅B pathways (Hwang et al., 2012). Further, studies with mammals showed a direct involvement of type I IFN in TLR3 induction, suggesting that TLR3 is an ISG and up-regulated in a positive feedback fashion (Tanabe et al., 2003). Collectively, the second expression wave in poly I:C-injected S. maximus might be a result of a positive loop. It was not observed, however, in the virus-injected group. It could be interpreted by a short time-course study or a weak positive feedback signal produced by TRBIV infection that was proved to inefficiently induce Mx in S. maximus (Hu et al., 2011a, b). In summary, the structure and expression pattern of a Tlr3 homologue in S. maximus and the structure of its promoter were characterized in this study. The smtlr3 transcripts were expressed ubiquitously and up-regulated by both poly I:C and DNA virus in the immune and non-immune organs. The strongest basal expression and highest inductive

© 2015 The Fisheries Society of the British Isles, Journal of Fish Biology 2015, 86, 431–447

A TL R3 H O M O L O G U E I N S C O P H T H A L M U S M A X I M U S

445

magnitude were both observed in the head kidney. Further, two waves of induced smtlr3 expressions were observed over a five day time course for poly I:C treatment. These findings may provide a new perspective for better understanding the functions of Smtlr3 in antiviral responses. The authors would like to thank J.-Y. Lin in our laboratory for her assistance during this study. This research was supported by grants from the National Basic Research Programme of China (2012CB114404), National Natural Science Foundation of China (30671604), Fundamental Research Funds for the Central Universities (201362024), Programme for New Century Excellent Talents in University of Ministry of Education of China (NCET-11-0467) and Shandong Provincial Natural Science Foundation (ZR2013CM045).

References Akira, S. & Takeda, K. (2004). Toll-like receptor signalling. Nature Reviews Immunology 4, 499–511. Alexopoulou, L., Holt, A. C., Medzhitov, R. & Flavell, R. A. (2001). Recognition of double-stranded RNA and activation of NF-kappaB by toll-like receptor 3. Nature 413, 732–738. Baoprasertkul, P., Peatman, E., Somridhivej, B. & Liu, Z. (2006). Toll-like receptor 3 and TICAM genes in catfish: species-specific expression profiles following infection with Edwardsiella ictaluri. Immunogenetics 58, 817–830. Bell, J. K., Mullen, G. E., Leifer, C. A., Mazzoni, A., Davies, D. R. & Segal, D. M. (2003). Leucine-rich repeats and pathogen recognition in toll-like receptors. Trends in Immunology 24, 528–533. Bell, J. K., Botos, I., Hall, P. R., Askins, J., Shiloach, J., Segal, D. M. & Davies, D. R. (2005). The molecular structure of the toll-like receptor 3 ligand-binding domain. Proceedings of the National Academy of Sciences of the United States of America 102, 10976–10980. Bell, J. K., Askins, J., Hall, P. R., Davies, D. R. & Segal, D. M. (2006). The dsRNA binding site of human toll-like receptor 3. Proceedings of the National Academy of Sciences of the United States of America 103, 8792–8797. Bilodeau, A. L. & Waldbieser, G. C. (2005). Activation of TLR3 and TLR5 in channel catfish exposed to virulent Edwardsiella ictaluri. Developmental and Comparative Immunology 29, 713–721. Breathnach, R. & Chambon, P. (1981). Organization and expression of eukaryotic split genes coding for proteins. Annual Review of Biochemistry 50, 349–383. Fan, S., Chen, S., Liu, Y., Lin, Y., Liu, H., Guo, L., Lin, B., Huang, S. & Xu, A. (2008). Zebrafish TRIF, a Golgi-localized protein, participates in IFN induction and NF-𝜅B activation. Journal of Immunology 180, 5373–5383. Funami, K., Matsumoto, M., Oshiumi, H., Akazawa, T., Yamamoto, A. & Seya, T. (2004). The cytoplasmic ‘linker region’ in toll-like receptor 3 controls receptor localization and signaling. International Immunology 16, 1143–1154. Gay, N. J., Gangloff, M. & Weber, A. N. (2006). Toll-like receptors as molecular switches. Nature Reviews Immunology 6, 693–698. Heinz, S., Haehnel, V., Karaghiosoff, M., Schwarzfischer, L., Müller, M., Krause, S. W. & Rehli, M. (2003). Species-specific regulation of toll-like receptor 3 genes in men and mice. Journal of Biological Chemistry 278, 21502–21509. Hu, G. B., Xia, J., Lou, H. M., Chen, X. L., Li, J. & Liu, Q. M. (2011a). An IRF-3 homolog that is up-regulated by DNA virus and poly I:C in turbot, Scophthalmus maximus. Fish and Shellfish Immunology 31, 1224–1231. Hu, G. B., Xia, J., Lou, H. M., Liu, Q. M., Lin, J. Y., Yin, X. Y. & Dong, X. Z. (2011b). Cloning and expression analysis of interferon regulatory factor 7 (IRF-7) in turbot, Scophthalmus maximus. Developmental and Comparative Immunology 35, 416–420. Huang, X.-N., Wang, Z.-Y. & Yao, C.-L. (2011). Characterization of toll-like receptor 3 gene in large yellow croaker, Pseudosciaena crocea. Fish and Shellfish Immunology 31, 98–106.

© 2015 The Fisheries Society of the British Isles, Journal of Fish Biology 2015, 86, 431–447

446

G .- B . H U E T A L.

Hwang, S. D., Ohtani, M., Hikima, J.-I., Jung, T. S., Kondo, H., Hirono, I. & Aoki, T. (2012). Molecular cloning and characterization of Toll-like receptor 3 in Japanese flounder, Paralichthys olivaceus. Developmental and Comparative Immunology 37, 87–96. Liu, L., Botos, I., Wang, Y., Leonard, J. N., Shiloach, J., Segal, D. M. & Davies, D. R. (2008). Structural basis of toll-like receptor 3 signaling with double-stranded RNA. Science 320, 379–381. Livak, K. J. & Schmittgen, T. D. (2001). Analysis of relative gene expression data using real-time quantitative PCR and the 2−ΔΔCT method. Methods 25, 402–408. Matsuo, A., Oshiumi, H., Tsujita, T., Mitani, H., Kasai, H., Yoshimizu, M., Matsumoto, M. & Seya, T. (2008). Teleost TLR22 recognizes RNA duplex to induce IFN and protect cells from birnaviruses. Journal of Immunology 181, 3474–3485. Meijer, A. H., Gabby Krens, S., Medina Rodriguez, I. A., He, S., Bitter, W., Ewa, S.-J. B. & Spaink, H. P. (2004). Expression analysis of the toll-like receptor and TIR domain adaptor families of zebrafish. Molecular Immunology 40, 773–783. Oshiumi, H., Tsujita, T., Shida, K., Matsumoto, M., Ikeo, K. & Seya, T. (2003). Prediction of the prototype of the human toll-like receptor gene family from the pufferfish, Fugu rubripes, genome. Immunogenetics 54, 791–800. Phelan, P. E., Mellon, M. T. & Kim, C. H. (2005). Functional characterization of full-length TLR3, IRAK-4, and TRAF6 in zebrafish (Danio rerio). Molecular Immunology 42, 1057–1071. Rebl, A., Goldammer, T. & Seyfert, H.-M. (2010). Toll-like receptor signaling in bony fish. Veterinary Immunology and Immunopathology 134, 139–150. Reed, L. J. & Muench, H. (1938). A simple method of estimating fifty per cent endpoints. American Journal of Epidemiology 27, 493–497. Rehli, M. (2002). Of mice and men: species variation of toll-like receptor expression. Trends in Immunology 23, 375–378. Rodriguez, M., Wiens, G., Purcell, M. & Palti, Y. (2005). Characterization of toll-like receptor 3 gene in rainbow trout (Oncorhynchus mykiss). Immunogenetics 57, 510–519. Sarkar, S. N., Smith, H. L., Rowe, T. M. & Sen, G. C. (2003). Double-stranded RNA signaling by toll-like receptor 3 requires specific tyrosine residues in its cytoplasmic domain. Journal of Biological Chemistry 278, 4393–4396. Sarkar, S. N., Peters, K. L., Elco, C. P., Sakamoto, S., Pal, S. & Sen, G. C. (2004). Novel roles of TLR3 tyrosine phosphorylation and PI3 kinase in double-stranded RNA signaling. Nature Structural and Molecular Biology 11, 1060–1067. Shi, C.-Y., Wang, Y.-G., Yang, S.-L., Huang, J. & Wang, Q.-Y. (2004). The first report of an iridovirus-like agent infection in farmed turbot, Scophthalmus maximus, in China. Aquaculture 236, 11–25. Su, J., Zhu, Z., Wang, Y., Zou, J. & Hu, W. (2008). Toll-like receptor 3 regulates Mx expression in rare minnow Gobiocypris rarus after viral infection. Immunogenetics 60, 195–205. Su, J., Jang, S., Yang, C., Wang, Y. & Zhu, Z. (2009). Genomic organization and expression analysis of toll-like receptor 3 in grass carp (Ctenopharyngodon idella). Fish and Shellfish Immunology 27, 433–439. Sullivan, C., Postlethwait, J. H., Lage, C. R., Millard, P. J. & Kim, C. H. (2007). Evidence for evolving toll-IL-1 receptor-containing adaptor molecule function in vertebrates. Journal of Immunology 178, 4517–4527. Tabeta, K., Georgel, P., Janssen, E., Du, X., Hoebe, K., Crozat, K., Mudd, S., Shamel, L., Sovath, S., Goode, J., Alexopoulou, L., Flavell, R. . A. & Beutler, B. (2004). Toll-like receptors 9 and 3 as essential components of innate immune defense against mouse cytomegalovirus infection. Proceedings of the National Academy of Sciences of the United States of America 101, 3516–3521. Tanabe, M., Kurita-Taniguchi, M., Takeuchi, K., Takeda, M., Ayata, M., Ogura, H., Matsumoto, M. & Seya, T. (2003). Mechanism of up-regulation of human toll-like receptor 3 secondary to infection of measles virus-attenuated strains. Biochemical and Biophysical Research Communications 311, 39–48. Wei, T., Gong, J., Rössle, S. C., Jamitzky, F., Heckl, W. M. & Stark, R. W. (2011). A leucine-rich repeat assembly approach for homology modeling of the human TLR5-10 and mouse TLR11-13 ectodomains. Journal of Molecular Modeling 17, 27–36.

© 2015 The Fisheries Society of the British Isles, Journal of Fish Biology 2015, 86, 431–447

A TL R3 H O M O L O G U E I N S C O P H T H A L M U S M A X I M U S

447

Werling, D. & Jungi, T. W. (2003). TOLL-like receptors linking innate and adaptive immune response. Veterinary Immunology and Immunopathology 91, 1–12. Yang, C. & Su, J. (2010). Molecular identification and expression analysis of toll-like receptor 3 in common carp Cyprinus carpio. Journal of Fish Biology 76, 1926–1939.

Electronic Reference Palumbi, S., Martin, A., Romano, S., McMillan, W., Stice, L. & Grabowski, G. (1991). The Simple Fool’s Guide to PCR, Version 2.0 (Privately Published Document Compiled by S. Palumbi). Honolulu, HI: Department of Zoology, University of Hawaii. Available at palumbi.stanford.edu/SimpleFoolsMaster.pdf

© 2015 The Fisheries Society of the British Isles, Journal of Fish Biology 2015, 86, 431–447

A toll-like receptor 3 homologue that is up-regulated by poly I:C and DNA virus in turbot Scophthalmus maximus.

In this study, the gene and promoter sequences of turbot Scophthalmus maximus (Sm) toll-like receptor 3 (Tlr3) were cloned and its mRNA tissue distrib...
1MB Sizes 0 Downloads 9 Views