Transcriptome analysis of orange-spotted grouper (Epinephelus coioides) spleen in response to Singapore grouper iridovirus

Abstract

BackgroundOrange-spotted grouper (Epinephelus coioides) is an economically important marine fish cultured in China and Southeast Asian countries. The emergence of infectious viral diseases, including iridovirus and betanodavirus, have severely affected food products based on this species, causing heavy economic losses. Limited available information on the genomics of E. coioides has hampered the understanding of the molecular mechanisms that underlie host-virus interactions. In this study, we used a 454 pyrosequencing method to investigate differentially-expressed genes in the spleen of the E. coioides infected with Singapore grouper iridovirus (SGIV).ResultsUsing 454 pyrosequencing, we obtained abundant high-quality ESTs from two spleen-complementary DNA libraries which were constructed from SGIV-infected (V) and PBS-injected fish (used as a control: C). A total of 407,027 and 421,141 ESTs were produced in control and SGIV infected libraries, respectively. Among the assembled ESTs, 9,616 (C) and 10,426 (V) ESTs were successfully matched against known genes in the NCBI non-redundant (nr) database with a cut-off E-value above 10-5. Gene ontology (GO) analysis indicated that "cell part", "cellular process" and "binding" represented the largest category. Among the 25 clusters of orthologous group (COG) categories, the cluster for "translation, ribosomal structure and biogenesis" represented the largest group in the control (185 ESTs) and infected (172 ESTs) libraries. Further KEGG analysis revealed that pathways, including cellular metabolism and intracellular immune signaling, existed in the control and infected libraries. Comparative expression analysis indicated that certain genes associated with mitogen-activated protein kinase (MAPK), chemokine, toll-like receptor and RIG-I signaling pathway were alternated in response to SGIV infection. Moreover, changes in the pattern of gene expression were validated by qRT-PCR, including cytokines, cytokine receptors, and transcription factors, apoptosis-associated genes, and interferon related genes.ConclusionThis study provided abundant ESTs that could contribute greatly to disclosing novel genes in marine fish. Furthermore, the alterations of predicted gene expression patterns reflected possible responses of these fish to the virus infection. Taken together, our data not only provided new information for identification of novel genes from marine vertebrates, but also shed new light on the understanding of defense mechanisms of marine fish to viral pathogens.

De novo assembly and characterization of the spleen transcriptome of common carp (Cyprinus carpio) using Illumina paired-end sequencing

“…Previous studies in other fish species have reported changes in the spleen transcriptome profile following exposure to antigens and chemicals, as well as temperature stress, thereby providing insight into immune-relevant genes and pathways. Examples of these species are the zebrafish (Danio rerio) [5], orange-spotted grouper (Epinephelus coioides) [6], large yellow croaker (Pseudosciaena crocea) [7,8], Atlantic cod (Gadus morhua) [9e11], Asian seabass (Lates calcarifer) [12], and channel catfish (Ictalurus punctatus) [13]. However, current knowledge regarding fish spleen is still fragmentary, and very little is known about the molecular processes in the fish spleen during normal development.…”

“…In recent years, high-throughput transcriptome sequencing has facilitated the functional genomic study of fish and has provided insights into the immunogenetics of fish. For example, previous studies have discovered the immune-relevant genes following exposure to pathogenic microorganisms through de novo transcriptome sequencing in several aquaculture fish species, including orangespotted grouper (E. coioides) [6], large yellow croaker (P. crocea) [7,8], turbot (Scophthalmus maximus) [17], Japanese sea bass (Lateolabrax japonicus) [18] and rohu carp (Labeo rohita, Hamilton) [19]. However, to our knowledge, the de novo sequencing of the spleen transcriptome of fish has been limited to only a few species, and this has hindered the progress of immunological research.…”

Contrasted Innate Responses to Two Viruses in Zebrafish: Insights into the Ancestral Repertoire of Vertebrate IFN-Stimulated Genes

“…Large-scale analyses of transcriptome response to viral infection have been carried out in a number of other fish species, including Atlantic salmon head kidney cell response to SAV-1 (12), Atlantic salmon heart response to piscine myocarditis virus (13), orange-spotted grouper spleen response to Singapore grouper iridovirus (14), response of the grouper kidney cell line infected by betanodavirus (15), and Atlantic cod brain response to nervous necrosis virus (16). These studies generally addressed the transcriptional response of a particular tissue after infection with a given virus, and resulted in overlapping but largely distinct sets of modulated genes.…”

Characterization of c-Jun from orange-spotted grouper, Epinephelus coioides involved in SGIV infection

“…The primers used for RACE PCR (Table 1) were designed based on the identified expressed sequence tag (EST) sequence from the transcriptome library established in our laboratory (Accession No. SRA040065.1) [28], In detail, the gene specific primer c-Jun 5 0 NGSP1, c-Jun 3 0 GSP1 (Table 1) and UPM (supplied by the kit) were used for the first round PCR. The PCR was conducted at 94 C for 3 min, followed by 30 cycles of 94 C for 30 s, 68 C for 30 s and 72 C for 50 s. The product of the first round PCR was diluted 10 times and then used as the template for the nested PCR.…”