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.