Blast2Fish: a reference-based annotation web tool for transcriptome analysis of non-model teleost fish.

BACKGROUND: Transcriptome analysis by next-generation sequencing has become a popular technique in recent years. This approach is quite suitable for non-model organism study, as de novo assembly is independent of prior genomic sequences of organisms. De novo sequencing has benefited many studies on commercially important fish species. However, to understand the functions of these assembled sequences, they still need to be annotated with existing sequence databases. By combining Basic Local Alignment Search Tool (BLAST) and Gene Ontology analysis, we were able to identify homologous sequences of assembled sequences and describe their characteristics using pre-defined tags for each gene, though the above conventional annotation results obtained for non-model assembled sequences was still associated with a lack of pre-defined tags and poorly documented records in the database. RESULTS: We introduced Blast2Fish, a novel approach for performing functional enrichment analysis on non-model teleost fish transcriptome data. The Blast2Fish pipeline was designed to be a reference-based enrichment method. Instead of annotating the BLAST single top hit by a pre-defined gene-to-tag database, we included 500 hits to search related PubMed articles and parse biological terms. These descriptive terms were then sorted and recorded as annotations for the query. The results showed that Blast2Fish was capable of providing meaningful annotations on immunology topics for non-model fish transcriptome analysis. CONCLUSION: Blast2Fish provides a novel approach for annotating sequences of non-model fish. The reference-based strategy allows annotation to be performed without pre-defined tags for each gene. This method strongly benefits non-model teleost fish studies for gene functional enrichment analysis.

Transcriptome profiling analysis of grouper during nervous necrosis virus persistent infection.

Nervous necrosis virus (NNV) infection has been considered a serious disease in farmed grouper. Particularly, the persistent infection model conducts the grouper into a carrier state that continues to spread the virus through spawning. This particular model makes disease control more difficult in the aquaculture industry. In the present study, we used RNA-Seq, a high-throughput method based on next-generation sequencing, to profile the expression of genes during the period of NNV persistent infection. We evaluated the transcriptomic changes in the brain tissue of grouper. The inactivated-NNV vaccine was used as a comparison group. Based on the differentially expressed genes, highly immune cell active signaling and surface receptor expression were triggered during persistent infection. The interferon-induced response was also highly expressed in the infected brain tissue. However, critical negative regulatory factors of T-cells, such as PD-L1 and LAG3, were up-regulated. The present transcriptome study revealed a comprehensive view of the state of NNV persistent infection and provided insights into the state of impaired NNV clearance in the grouper.

Interferon regulatory factor-1 (IRF-1) is involved in the induction of phosphatidylserine receptor (PSR) in response to dsRNA virus infection and contributes to apoptotic cell clearance in CHSE-214 cell.

The phosphatidylserine receptor (PSR) recognizes a surface marker on apoptotic cells and initiates engulfment. This receptor is important for effective apoptotic cell clearance and maintains normal tissue homeostasis and regulation of the immune response. However, the regulation of PSR expression remains poorly understood. In this study, we determined that interferon regulatory factor-1 (IRF-1) was dramatically upregulated upon viral infection in the fish cell. We observed apoptosis in virus-infected cells and found that both PSR and IRF-1 increased simultaneously. Based on a bioinformatics promoter assay, IRF-1 binding sites were identified in the PSR promoter. Compared to normal viral infection, we found that PSR expression was delayed, viral replication was increased and virus-induced apoptosis was inhibited following IRF-1 suppression with morpholino oligonucleotides. A luciferase assay to analyze promoter activity revealed a decreasing trend after the deletion of the IRF-1 binding site on PSR promoter. The results of this study indicated that infectious pancreatic necrosis virus (IPNV) infection induced both the apoptotic and interferon (IFN) pathways, and IRF-1 was involved in regulating PSR expression to induce anti-viral effects. Therefore, this work suggests that PSR expression in salmonid cells during IPNV infection is activated when IRF-1 binds the PSR promoter. This is the first report to show the potential role of IRF-1 in triggering the induction of apoptotic cell clearance-related genes during viral infection and demonstrates the extensive crosstalk between the apoptotic and innate immune response pathways.

Identification of the STAT1 gene and the characterisation of its immune response to immunostimulants, including nervous necrosis virus (NNV) infection, in Malabar grouper (Epinephelus malabaricus).

Signal Transducer and Activator of Transcription (STAT)-1 is an indispensable signal transduction protein that is involved in the interferon pathway. STAT-1 plays an important role in the innate immune response. The full-length cDNA of Malabar grouper (Epinephelus malabaricus) STAT-1, MgSTAT1, was cloned. Phylogenetic analysis was performed based on the amino acid sequence. Our results indicate that STAT1 is highly conserved with other vertebrates. We also report the expression of MgSTAT1 in different tissues treated with immune stimulants, including LPS, CpG ODN, and poly (I:C), in vivo. The expression of MgSTAT1 was significantly induced in the head kidney upon treatment with poly (I:C) compared to the control. Moreover, the results indicate that MgSTAT1 is up-regulated during nervous necrosis virus (NNV) infection. This study reveals that similar to the mammalian antiviral response, MgSTAT1 mediates the immune response in Malabar grouper.

A novel quantitative immunomagnetic reduction assay for Nervous necrosis virus.

Rapid, sensitive, and automatic detection platforms are among the major approaches of controlling viral diseases in aquaculture. An efficient detection platform permits the monitoring of pathogen spread and helps to enhance the economic benefits of commercial aquaculture. Nervous necrosis virus (NNV), the cause of viral encephalopathy and retinopathy, is among the most devastating aquaculture viruses that infect marine fish species worldwide. In the present study, a highly sensitive magnetoreduction assay was developed for detecting target biomolecules with a primary focus on NNV antigens. A standard curve of the different NNV concentrations that were isolated from infected Malabar grouper (Epinephelus malabaricus) was established before experiments were conducted. The test solution was prepared by homogeneous dispersion of magnetic nanoparticles coated with rabbit anti-NNV antibody. The magnetic nanoparticles in the solution were oscillated by magnetic interaction with multiple externally applied, alternating current magnetic fields. The assay's limit of detection was approximately 2 × 10(1) TCID(50)/ml for NNV. Moreover, the immunomagnetic reduction readings for other aquatic viruses (i.e., 1 × 10(7) TCID(50)/ml for Infectious pancreatic necrosis virus and 1 × 10(6.5) TCID(50)/ml for grouper iridovirus) were below the background noise in the NNV solution, demonstrating the specificity of the new detection platform.