Characterization of the transcriptomic response to infection is an effective approach to understanding the immune mechanisms. Herein we challenged grass carp (Ctenopharyngodon idella) with grass carp reovirus (GCRV) and sequenced four cDNA libraries obtained from head-kidney and spleen by using Illumina Miseq. As a result, we gained a total of 21.52 Gb clean data with 107.96 million reads, and de novo assembled 55,199 unigenes with an average length of 1,470 bp. Comparative transcriptome analysis reveals that 217 unigenes are differentially expressed (fold-change of at least 4) between resistant and susceptible fish in both head-kidney and spleen, and of which 36 unigenes were validated by RT-qPCR experiment. The expression profile of immune-related genes demonstrates that the immune response of spleen is more intense than that of head-kidney. Remarkably, 11,811 unigenes contain multiple transcripts, of which 322 unigenes possess notably differentially expressed transcripts between the four transcriptomic datasets. Furthermore, the splicing transcripts of IL-12p40 and IL-1R1 are firstly found to play diverse roles in the antiviral response of fishes. This study provides a complete transcriptome dataset of C. idella, which is valuable for the studies of immune complexity and, moreover, throws light on the regulatory role of AS in antiviral immunity.

High mobility group box (HMGB) proteins, a family of chromatin-associated nuclear proteins, exert amazingly multifaceted roles in the immune system in mammals. Thus far, little is known about the nucleocytpolasmic distribution of HMGBs in teleosts. Present study systematically investigated the dynamic localization of all the six HMGB proteins in Ctenopharyngodon idella kidney (CIK) cells. Under basal condition, all HMGBs exclusively located in nucleus. Grass carp reovirus (GCRV), polyinosinic–polycytidylic potassium salt (poly(I:C)) and lipopolysaccharide (LPS) challenge evoked nuclear export of HMGBs in various degrees: GCRV challenge induced the most nuclear export of CiHMGB2b, and the most nucleocytoplasmic shuttling of CiHMGB1b was evoked by poly(I:C) and LPS. Overall, nucleocytoplasmic shuttling of CiHMGB2a and CiHMGB3b was rarely induced by those challenges. Dynamic imaging uncovered that nucleocytoplasmic relocation of CiHMGB2b induced by GCRV occurred in cells undergoing three physiological states: karyotheca rupture, apoptosis and proliferation. Western bolt examined HMGBs-EGFP fusion proteins in whole cell lysates, cytosol, nuclear fractions and culture medium. Further study demonstrated the nucleus retention of N-terminal HMG-boxes and nucleocytoplasmic distribution of C-terminal acidic tail. Comparative analyses of the dynamic relocation of full-length, truncated or chimeric HMGBs confirmed that intra-molecular interaction between HMG-boxes and C-tail domains mediated nucleocytoplasmic translocation of HMGBs. These results firstly provide overall understanding of subcellular localization of HMGBs, and also preliminary uncover the induction mechanism of nucleocytoplasmic translocation of HMGBs by GCRV challenge, which lay a foundation for the further researches on interaction among pathogens-HMGBs-pattern recognition receptors (PRRs) in innate immunity of teleosts.

Melanoma differentiation-associated gene 5 (MDA5) plays a crucial role in recognizing intracellular viral infection, activating the interferon regulatory factor pathways as well as inducing antiviral response. While the antiviral regulatory mechanism of MDA5 remains unclear. In the present study, CiMDA5 (Ctenopharyngodon idella MDA5) against grass carp reovirus (GCRV) would be initially revealed from the perspective of DNA methylation, a pivotal epigenetic modification. Two CpG islands (CGIs) were predicted located in the first exon of CiMDA5, of which the first CpG island was 427 bp in length possessed 29 candidate CpG loci and 34 CpA loci, and the second one was 130 bp in length involving 7 CpG loci as well as 10 CpA loci. By bisulfite sequencing PCR (BSP), the methylation statuses were detected in spleen of 70 individuals divided into resistant/susceptible groups post challenge experiment, and the resistance-association analysis was performed with Chi-square test. Quantitative real-time RT-PCR (qRT-PCR) was carried out to explore the relationship between DNA methylation and gene expression in CiMDA5. Results indicated that the methylation levels of CpA/CpG sites at +200, +202, +204, +207 nt, which consisted of a putative densely methylated element (DME), were significantly higher in the susceptible group than those in the resistant group. Meanwhile, the average transcription of CiMDA5 was down-regulated in the susceptible individuals compared with the resistant individuals. Evidently, the DNA methylation may be the negative modulator of CiMDA5 antiviral expression. Collectively, the methylation levels of CiMDA5 were demonstrated the tight association with the resistance against GCRV and the negative-regulated roles in mRNA expression. This study first discovered the resistance-associated gene modulated by DNA methylation in teleost, preliminary revealed the underlying regulatory mechanism of CiMDA5 transcription against GCRV as well as laid a theoretical foundation on molecular nosogenesis of hemorragic diseases in C. idella.

Global fish production from aquaculture has rapidly grown over the past decades, and grass carp shares the largest portion. However, hemorrhagic disease caused by grass carp reovirus (GCRV) results in tremendous loss of grass carp (Ctenopharyngodon idella) industry. During the past years, development of molecular biology and cellular biology technologies has promoted significant advances in the understanding of the pathogen and the immune system. Immunoprophylaxis based on stimulation of the immune system of fish has also got some achievements. In this review, authors summarize the recent progresses in basic researches on GCRV; viral nucleic acid sensors, high-mobility group box proteins (HMGBs); pattern recognition receptors (PRRs), Toll-like receptors (TLRs) and retinoic acid inducible gene I- (RIG-I-) like receptors (RLRs); antiviral immune responses induced by PRRs-mediated signaling cascades of type I interferon (IFN-I) and IFN-stimulated genes (ISGs) activation. The present review also notices the potential applications of molecule genetic markers. Additionally, authors discuss the current preventive and therapeutic strategies (vaccines, RNAi, and prevention medicine) and highlight the importance of innate immunity in long term control for grass carp hemorrhagic disease.

ADAR (adenosine deaminase acting on RNA) is an RNA editing enzyme that targets both coding and noncoding dsRNAs (double stranded RNAs) and converts adenosine to inosine, which is read by translation machinery and by polymerases during RNA-dependent RNA replication as if it is guanosine. This editing is a widespread post-transcriptional modification event in animals. In this study, we identified the full-length cDNA sequence of Ctenopharyngodon idella ADAR1 (designated as CiADAR1) and detected the mRNA expression patterns in response to dsRNA (polyinosinic-polycytidylic acid sodium salt, poly(I:C)) and grass carp reovirus (GCRV). CiADAR1 is a large gene in size, consisting of 4833 nucleotides encoding a protein of 1392 amino acids. The deduced amino acid sequence contains seven putative domains: one proline-rich region (Pro-R), two Z-DNA-binding domains (Zalpha), three dsRNA binding motifs (DSRM) and one tRNA-specific and dsRNA adenosine deaminase domain (ADEAMc). It is most homologous to Danio rerio ADAR (E-value = 0.0, identities = 80% (1110/1395)), also close homology to Homo sapiens ADAR1 (E-value = 0.0, identities = (47%) 530/1122). CiADAR1 mRNA was investigated in fifteen tissues, and was low expressed in muscle and head kidney tissues, high in blood and spleen tissues by quantitative real-time RT-PCR (qRT-PCR). mRNA expressions of CiADAR1 were significantly up-regulated and reached peak at 24 h post GCRV challenge in vivo and in vitro (P < 0.05). After poly(I:C) stimulation at different concentrations, CiADAR1 transcripts were rapidly and significantly up-regulated and recovered in dose-dependent and time-dependent manners (P < 0.05). The results indicate CiADAR1 was implicated in the antiviral immune response and laid the foundation for further studies on functions and mechanisms of RNA editing in fishes.

Stimulator of interferon gene (STING), an important adapter responsible for RLR pathway, plays a pivotal role in both viral RNA- and DNA-triggered induction of IFNs in mammals. To understand the roles of STING in piscine immune system, STING gene (CiSTING) was identified from grass carp (Ctenopharyngodon idella). The genomic sequence of CiSTING was of 8548 base pairs (bp), including 899 bp 5′ flank region, 7 exons and 6 introns. Promoter region was predicted and promoter activity was verified. The CiSTING cDNA was of 1358 bp with an open reading frame of 1185 bp, encoding a polypeptide of 394 amino acids with a signal peptide and three transmembrane motifs in the N-terminal region. mRNA expression of CiSTING was widespread in fifteen tissues investigated, and was up-regulated by GCRV in vivo and in vitro. Meanwhile, the transcription of CiSTING was inhibited at early stage, and then up-regulated at late phase upon poly(I:C) or PGN stimulation in vitro. Interestingly, CiSTING had little impact on LPS in vitro. In CiSTING over-expression cells, CiTBK1, CiIRF3 and CiIRF7 were significantly up-regulated post GCRV or viral/bacterial PAMPs stimulation. In addition, post GCRV or PGN stimulation, the transcription of CiIFN-I was remarkably inhibited while CiMx1 was up-regulated; as for poly(I:C) stimulation, mRNA expressions of CiIFN-I and CiMx1 were inhibited at early stage while enhanced at late phrase; after LPS stimulation, both CiIFN-I and CiMx1 were inhibited. Furthermore, antiviral activity of CiSTING was manifested by the inhibition of GCRV yield. Taken together, these results demonstrated that CiSTING may be involved in board innate immune responses via the TBK1-IRF3/IRF7 cascade, responding to not only dsRNA analogue in an IFN-dependent pathway, but also virus and bacterial PAMPs in an IFN-independent pathway. This study provided novel insights into the essential role of STING in innate immunity.

Toll-like receptor 7 (TLR7) gene was identified and characterised from grass carp Ctenopharyngodon idella (designated as CiTLR7), and the mRNA expression profiles were examined in vivo and in vitro. The CiTLR7 genomic sequence consists of 4276 nucleotides (nts), including two exons and one intron. The full length of CiTLR7 cDNA sequence is 3354 nts with the longest open reading frame (ORF) of 3156 nts encoding a peptide of 1051 amino acids. CiTLR7 mRNA was high expression in spleen, skin and heart, and low expression in hepatopancreas, muscle, head kidney and trunk kidney in healthy fish. The expression of CiTLR7 was rapidly and significantly up-regulated at 6 h after grass carp reovirus (GCRV) injection (72.91 fold, P < 0.05), and recovered to the original level at 24 h post-injection in spleen. The CiTLR7 transcript was rapidly and significantly down-regulated at 6 h time point (0.32 fold, P < 0.05) and retrieved the normal level at 72 h post-injection in hepatopancreas. The CiTLR7 transcripts were rapidly and significantly inhibited at 2 h post GCRV infection in CIK (C. idella kidney) cell line (0.62 fold, P < 0.05), and were rapidly and significantly elevated by the stimulation of the synthetic double-stranded RNA polyriboinosinic polyribocytidylic acid sodium salt (poly(I:C)) in CIK in a dose- and time-dependent manners (P < 0.05). The results imply that CiTLR7 involves in the responses to double stranded RNA and virus infection.

Toll/interleukin-1 receptor (TIR) domain containing adapter inducing interferon-β (TRIF) is an adapter in responding to activation of some toll-like receptors (TLRs), which provides early clearance of viral and bacterial pathogens. Here we identified and characterized a full-length genomic sequence of TRIF gene from grass carp Ctenopharyngodon idella (designated as CiTRIF). CiTRIF genomic sequence consists of 3534 base pairs (bp), containing 5’ flank sequence (496 bp) and unique intron (815 bp). The full-length cDNA sequence is 2241 bp, including 5’ untranslated region (UTR) of 352 bp, 3’ UTR of 209 bp, and an open reading frame of 1680 bp encoding a polypeptide of 559 amino acids with an estimated molecular weight of 62.643 kDa and a predicted isoelectric point of 5.71. The deduced amino acid sequence just contains TIR domain, and is most similar to the zebrafish (Danio rerio) TRIF sequence with an identity of 64%. CiTRIF exhibits sequence divergence from its orthologs. Promoter region was predicted and promoter activity was verified. mRNA expression of CiTRIF gene is widespread in fifteen tissues investigated, highly in foregut and skin physiological immune barrier. The transcripts of CiTRIF were significantly and rapidly induced in spleen and head kidney tissues at early stage post grass carp reovirus (GCRV) challenge. The modulations are significant but mild in CIK (C. idella kidney) cells post GCRV infection or poly(I:C) stimulation. The over-expression vector was constructed and transfected into CIK cell line to get stably expressing recombinant proteins. In CiTRIF transfected cells, mRNA expressions of CiTRIF, CiRIG-I, CiIRF7 and CiIFN-I were up-regulated. After GCRV infection, the transcripts of CiTRIF, CiRIG-I, CiIRF7 and CiIFN-I fell a little bit after a rapidly and strongly rise. In CiTRIF over-expression cells, virus load and titer were significantly lower than those in controls post GCRV challenge, and virus replication was inhibited obviously. The results indicate that the novel TRIF gene from grass carp plays important roles in modulating antiviral innate immune responses, and serve the further functional studies on TRIF gene in teleosts and immune evolution.

High mobility group box 3 (HMGB3) protein is a universal sentinel in the activation of innate antiviral immune responses in mammalian cells of limited tissues. However, the underlying immune functions of HMGB3 responding to viruses and viral/bacterial pathogen-associated molecular patterns (PAMPs) are still unknown in teleosts. In the present study, two novel homologs of grass carp (Ctenopharyngodon idella) HMGB3 (designated as CiHMGB3a and CiHMGB3b) were identified and characterized. Quantitative RT-PCR analysis showed that CiHMGB3a and CiHMGB3b were widely expressed in tissues. The mRNA expressions of CiHMGB3a and CiHMGB3b were induced by grass carp reovirus (GCRV) challenges both in tissues and in cells, and CiHMGB3a played a more active role in antiviral immune responses. Viral PAMP stimulation evidenced that CiHMGB3a and CiHMGB3b mediated immune responses in CIK (C. idella kidney) cells. Interestingly, CiHMGB3a had little impact on bacterial PAMPs (LPS and PGN), whereas CiHMGB3b was critical responding to bacterial PAMPs stimulation. In overexpressions of CiHMGB3a and CiHMGB3b cells, the transcriptional levels of CiHMGB3a, CiHMGB3b, CiTRIF, CiIPS-1, CiIFN-I and CiMx1 were remarkably induced. In addition, CiMyD88 had vital impact on antiviral signaling channels in overexpression of CiHMGB3b cells. Furthermore, 96-well plate staining assay, virus titer test and GCRV quantitative analysis collectively indicated CiHMGB3a and CiHMGB3b exhibited substantial antiviral activity. These results suggest that CiHMGB3a and CiHMGB3b exert important functions in antiviral immune responses by TLRs and RLRs signaling pathways. Taken together, current study provides the first evidence that HMGB3 participates in broad antiviral and antibacterial immune responses in teleosts.

High-mobility group box 2 (HMGB2) protein is a chromatin-associated nonhistone protein, involved in transcriptional regulation and nucleic-acid-mediated innate immune responses in mammalian. However, the function of piscine HMGB2 in innate immune responses is still unknown. In the present study, two HMGB2 homologue genes (CiHMGB2a, CiHMGB2b) were identified and characterized in grass carp (Ctenopharyngodon idella). Both CiHMGB2a and CiHMGB2b genes encode proteins with 213 amino acids, sharing 71.4% identities and containing two basic HMG boxes and an acidic tail. The deduced protein sequences showed the most identities to HMGB2a (93%) and HMGB2b (86.4%) of zebrafish (Danio rerio), respectively. Quantitative real-time RT-PCR (qRT-PCR) analysis showed that CiHMGB2a and CiHMGB2b were constitutively expressed in all the 15 tested tissues. Post grass carp reovirus (GCRV) infection, mRNA levels of CiHMGB2a and CiHMGB2b were strongly up-regulated in spleen and head kidney and mildly modulated in C. idella kidney (CIK) cells. Meanwhile, mRNA expressions of CiHMGB2a and CiHMGB2b were significantly regulated by viral pathogen associated molecular patterns (PAMPs) polyinosinic-polycytidylic potassium salt (poly(I:C)) and bacterial PAMPs lipopolysaccharide (LPS), peptidoglycan (PGN) challenge in CIK cells. In CiHMGB2a and CiHMGB2b over-expression cells, expressions of CiHMGB2a and CiHMGB2b facilitated each other; transcription levels of CiTRIF, CiMyD88, CiIPS-1, and CiMx1 were remarkably enhanced, whereas CiIFN-I was inhibited, compared with those in cells transfected with pCMV (control plasmid); after GCRV challenge, all those tested genes were up-regulated with divergent expression profiles. Antiviral activities of CiHMGB2a and CiHMGB2b were manifested by the delayed appearance of cytopathic effect (CPE) and inhibition of GCRV yield. All those results demonstrate that CiHMGB2a and CiHMGB2b not only mediate antiviral immune responses but also involve in responding to viral/ bacterial PAMPs challenge, which provides novel insights into the essential role of HMGB2 in innate immunity.