An increasing number of reports reveal that transcriptome sequencing of cDNA has became an efficient strategy for generating enormous sequences that represent expressed genes . Transcriptomes from a number of species including those from Drosophila melanogaster, yeast, Caenorhabditis elegans and various mammals and plants were carried out for different purposes [17–21]. However, genome and transcriptome data for many "lower" vertebrate species, particularly marine fishes, have not been disclosed. To our knowledge, a limited numbers of E. coioides genes were cloned and characterized, based on the bioinformatic analysis, including those involved in immune responses after pathogenic attack, growth and development [22–27]. Given that the spleen is one of the most important organs associated with immune responses in fish and is also the main target organ for SGIV infection, the transcriptome sequencing of the E. coioides spleen can be expected to provide a significant number of ESTs for marine fish immune responses and contribute to understanding iridovirus-host interactions .
After removal of overlapping sequences between the control and SGIV-infected libraries, we obtained 65374 non-redundant consensus sequences from E. coioides. With the exception of sequences related to cellular structure and metabolism, abundant sequences were found to be homologous to known immune-relevant genes in other species, based on the BLAST, Conserved Domain Database (CDD), and SWISS-PROT annotation [28–30]. More than 80 sequences shared homology to signaling molecules of the mammalian mitogen-activated protein kinase (MAPK) pathways, such as critical molecules associated with extracellular signal-regulated kinase (ERK), p38 MAPK, Ras, RSK2, MKK4, MKK7, ASK1, MEK1/2 and Raf1. The mammalian MAPK signaling pathway was activated during virus infection and contributed to virus replication [31–33]. Although the MAPK signaling molecules including ERK, c-Jun N-terminal kinase (JNK) and p38 MAPK were activated in the spleens of SGIV-infected fish (EAGS) cells, identifying the exact roles of these molecules during SGIV replication will benefit from the E. coioides EST information [34, 35]. With the exception of homologue components in the MAPK cascade, different members of interferon-related genes were obtained, including the interferon-induced protein viperin, the interferon-stimulated gene 15 (ISG15), interferon-induced protein 35 kD (IFP35), interferon-stimulated gene 56 (ISG56), and interferon regulatory factors (IRF-1, IRF-2, IRF-3, IRF-4, IRF-5, IRF-7, IRF-8 and IRF-9). Interferon-induced, or stimulated, genes were important for the resistance of the host to virus infection, including virus entry, replication and release [36–38]. The E. coioides IRF-1, IRF-2 and IRF-7 genes have been cloned and characterized and IRF-7 was confirmed as being vitally important for SGIV replication [39, 40]. Human ISG15 expression is strongly up-regulated during viral infections, such as human cytomegalovirus (HCMV) and herpes simplex virus (HSV), and ISG15 up-regulation was considered to be involved in different strategies relating to the antiviral response [41–44]. IFP35 and ISG56 were also involved in the cellular antiviral response against virus infection [38, 45]. A detailed investigation on the functions of E. coioides interferon-related genes during SGIV infection will contribute greatly to understanding how the SGIV exploited, or evaded, the host interferon immune response.
We also obtained sequences that shared homology to SGIV-encoded immune evasion genes, including lipopolysaccharide-induced tumor necrosis factor-α factor (LITAF), tumor necrosis factor receptor (TNFR), ubiquitin and Bcl-2 [46–48]. Iridovirus-encoded LITAF and Bcl-2 could mediate the fate of host cells by regulating apoptosis [47, 48]. It has been reported that many viral immune evasion genes are considered as "stolen" mimics from the host and such viruses may interfere with the host response by modulating or disrupting the function of corresponding host genes [49–51]. The discovery of these sequences will be helpful in studies on host-virus interactions. In addition, we also found that other molecules such as lectin, hepcidin, lysozyme and antimicrobial peptide are involved in immune responses. The functions of these genes during virus infection will be investigated in the further studies.
Based on results from exploratory statistical analysis, we identified genes that are up-regulated or down-regulated after SGIV infection. The present data from qRT-PCR analysis validated the hypothesis that expression of partial genes is regulated by SGIV infection, including cytokine, cytokine receptor and transcription factor, apoptosis-associated genes, interferon-related genes, and cytoskeleton genes. Previous studies indicated that the expression of different groups of genes relating to cellular structure, apoptosis, gene transcription and immune regulation were altered in response to virus infections or other stimuli [37, 52–54]. Further research into the roles of these differentially-expressed genes will contribute to an increased understanding of the critical events that take place during SGIV replication.