Expression, signal transduction, and function analysis of TIRAP and TRIF in Nile tilapia (Oreochromis niloticus).

Toll/interleukin 1 receptor domain-containing adaptor protein (TIRAP) and toll/interleukin 1 receptor-domain-containing adapter-inducing interferon-ß (TRIF) are crucial adaptors of signal transduction for the signaling pathways of toll-like receptors (TLRs). TIRAP and TRIF perform an essential function in an antimicrobial immune response; however, their function in Nile tilapia remains unknown. Herein, TIRAP and TRIF from Nile tilapia were identified and functionally characterized. Phylogenetic analysis showed that OnTIRAP and OnTRIF clustered with corresponding homologs from other fish species, with comparable gene structures to those of select vertebrate TIRAP and TRIF genes, respectively. The expression profiles of OnTIRAP and OnTRIF were broadly distributed in the ten tissues investigated, with high transcript levels noticed in immune organs. The transcription levels of OnTIRAP and OnTRIF were upregulated in response to bacterial and poly (I:C) challenges. GFP signals were only detected in the cytoplasmic region of fish cells transfected with OnTIRAP-GFP and OnTRIF-GFP expression plasmids. Moreover, overexpression of OnTIRAP and OnTRIF activated interferon-ß (IFN-ß) and activator protein 1 (AP1) reporters in HEK 293 cells. Activation of the nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) reporter was only observed in OnTRIF-overexpressing HEK 293 cells. Furthermore, the results of the co-immunoprecipitation analysis showed that OnTRIF, but not OnTIRAP, was recruited as an adaptor protein by OnTLR25. This study provides the first evidence on the functions of OnTIRAP and OnTRIF in the immune system of Nile tilapia against pathogens and may serve as the basis for further investigations on TLR signaling in fish.

Immunostimulatory effects of Sarcodia suiae water extracts on Nile tilapia Oreochromis niloticus and its resistance against Streptococcus agalactiae.

In this study, water extracts of the red seaweed Sarcodia suiae were obtained using solid-liquid extraction (SLE) or pressurized liquid extraction (PLE) methods. The extracts were used to investigate immunostimulatory activity by measuring the phagocytic activity of Nile tilapia (Oreochromis niloticus) hepatic and splenic macrophages and the tilapia head kidney (THK) cell line, and modulation of immune-related genes in primary head kidney (HK) cells and THK cells. At 10 µg/ml, both extracts promoted the proliferation of hepatic and splenic macrophages. Expression levels of proinflammatory cytokines (IL-1ß and IL-8), antimicrobial peptides (TP2 and TP4), and pattern recognition receptors (TLR5) were elevated in SLE extracts-treated primary HK leukocytes. Similarly, IL-1ß, IL-8, and TNFα expression was also induced by SLE extract in THK cells. Phagocytic activity in primary HK cells and THK cells was induced by SLE extract 12 h and 24 h post-stimulation, while PLE extract only induced phagocytic activity in THK cells at early time points. SLE extract (100 µg/g body weight) increased the expression of IL-1ß, IL-8, TNFα, TP2, TP4, TLR2 and TLR5 in the spleen and immunoprotective efficiency against Streptococcus agalactiae infection. Taken together, these results show that S. suiae can differentially stimulate the immune response of tilapia in vitro and in vivo and could potentially be used as an immunomodulator in tilapia culture.

Complete genome sequence and phylogenetic analysis of a novel aquareovirus isolated from a diseased marbled eel (Anguilla marmorata).

Marbled eel reovirus (MERV) is an aquareovirus (AQRV) isolated from diseased marbled eels (Anguilla marmorata) with petechial skin hemorrhage. In this study, we propagated MERV in a cell line derived from the brain of Aequidens rivulatus and purified viral particles by using a discontinuous cesium chloride gradient. Genomic RNA sequences were obtained through next-generation sequencing. MERV, similar to most other AQRVs, showed the presence of 11 double-stranded RNA segments encoding 12 proteins; however, the genome sequence displayed very little similarity to known AQRV sequences. Furthermore, the structural proteins of MERV were most closely related to American grass carp reovirus with sequence identity values of no more than 64.89%. Phylogenetic analysis based on the sequences of structural proteins indicated that MERV shows an evolutionary history between AQRV-B and -G, which belong to the saline and freshwater environment subgroups, respectively. We also observed that MERV showed a closer relationship to orthoreoviruses based on the protein sequences of NS38 and NS73. In summary, MERV is a novel AQRV that could be classified as a member of the new proposed AQRV species "Aquareovirus H". The taxonomic assignments and evolution of AQRVs thus warrant further investigation.

Persistent development of adomavirus and aquareovirus in a novel cell line from marbled eel with petechial skin haemorrhage.

In Taiwan, a petechial haemorrhage disease associated with mortality has affected marbled eels (Anguilla marmorata). The eels were revealed to be infected with adomavirus (MEAdoV, previously recognized as a polyoma-like virus). In this study, cell line DMEPF-5 was established from the pectoral fin of a diseased eel. DMEPF-5 was passaged >70 times and thoroughly proliferated in L-15 medium containing 2%-15% foetal bovine serum at 20-30°C. Transcripts of neural cell adhesion molecule 1 and nestin genes, and nucleic acids of MEAdoV and a novel reovirus (MERV) in the cells were demonstrated by reverse transcription-polymerase chain reaction analysis. Phylogenetic analysis revealed that the AdoV LO8 proteins mostly relate to adenovirus adenain, whereas MERV is close to American grass carp reovirus in Aquareovirus G, based on a partial VP2 nucleotide sequence. DMEPF-5 cells are susceptible to additional viral infection. Taken together, the marbled eels with the haemorrhagic disease have coinfection with MEAdoV and MERV, and the pathogenic role of MEAdoV and MERV warrants research. DMEPF-5 has gene expression associated with mesenchymal stem and progenitor cells and is the first cell line persistently infected with adomavirus and aquareovirus. DMEPF-5 can facilitate studies of such viruses and haemorrhagic disease.

Complete genome sequence and phylogenetic analysis of megalocytivirus RSIV-Ku: A natural recombination infectious spleen and kidney necrosis virus.

Megalocytiviruses are classified into three genotypes, infectious spleen and kidney necrosis virus (ISKNV), red seabream virus (RSIV), and turbo reddish body iridovirus (TRBIV), based on the major capsid protein and ATPase genes. However, only a few complete genome sequences have been obtained. This paper reports the complete genome sequence and phylogenetic analysis of an RSIV-Ku strain megalocytivirus. The genome sequence comprises 111,154 bp, has 132 putative open reading frames, and is homologous mostly to ISKNV, except for the sequence in the region 58981-66830, which is more closely related to that of the RSIV genotype. The results imply that RSIV-Ku is actually a natural recombinant virus.

Complete genome sequence and phylogenetic analyses of an aquabirnavirus isolated from a diseased marbled eel culture in Taiwan.

An aquabirnavirus was isolated from diseased marbled eels (Anguilla marmorata; MEIPNV1310) with gill haemorrhages and associated mortality. Its genome segment sequences were obtained through next-generation sequencing and compared with published aquabirnavirus sequences. The results indicated that the genome sequence of MEIPNV1310 contains segment A (3099 nucleotides) and segment B (2789 nucleotides). Phylogenetic analysis showed that MEIPNV1310 is closely related to the infectious pancreatic necrosis Ab strain within genogroup II. This genome sequence is beneficial for studying the geographic distribution and evolution of aquabirnaviruses.

Genome Sequence of a Marbled Eel Polyoma-Like Virus in Taiwan.

We report here the complete genome sequence of a virus isolated from a diseased marbled eel (Anguilla marmorata) in Taiwan. The virus has been characterized as being related to Japanese eel endothelial cell-infecting virus (JEECV), with a large T-antigen-like protein. The sequence of the marbled eel virus displays low homology to the JEECV.

Complete Genome Sequence of Herpesvirus anguillae Strain HVA980811 Isolated in Chiayi, Taiwan.

Herpesvirus anguillae (HVA), also known as anguillid herpesvirus 1 (AngHV1), is one of the relevant viruses in wild and cultured anguillid eels. Here, the complete genome sequence of strain HVA980811, isolated in Chiayi, Taiwan, is reported. The genotype of the eel herpesviruses in Taiwan is supposed to be identical to the Japanese AngHV1.

Complete Genome Sequence of a Giant Sea Perch Iridovirus in Kaohsiung, Taiwan.

We report here the complete genome sequence of a megalocytivirus strain, GSIV-K1, isolated from a farmed giant sea perch (Lates calcarifer) in Kaohsiung, Taiwan. GSIV-K1 causes mortality in farmed marine fish, including giant sea perch and groupers. The genome sequence is nearly identical to the genome of the orange-spotted grouper iridovirus.

Development and characterization of a cell line from tilapia head kidney with melanomacrophage characteristics.

A novel cell line THK, derived from the tilapia head kidney, was developed and characterized. The THK cell line comprised fibroblastoid cells that markedly proliferated in Leibovitz L-15 medium containing 2%-15% fetal bovine serum (FBS) at 20 °C-35 °C. Cell proliferation was dependent on the FBS concentration, and the optimal temperature for proliferation ranged between 25 °C and 30 °C. THK cells were characterized for the presence of phagocytic activity, acid phosphatase, alkaline phosphatase, α-naphthyl acetate esterase, lipofuscin, and tyrosinase. Transcripts of CD33, CD53, CD82, CD205, macrophage colony stimulating factor receptor, GATA2, and GATA3 that are specific for leucocytes or monocytes/macrophages or both were detected in the THK cells through PCR. However, THK cells lacked for CD83, a specific marker for dendritic cells. The results indicated that the fibroblastoid THK cells were melanomacrophage-related progenitors. PCR revealed that the THK cells exhibited the transcripts of toll-like receptor 1 (TLR1), TLR2, TLR3, and CD200, of which concern with immunity as well as the transcripts of vascular endothelial growth factor receptor 3, angiomotin, and angiopoietin-like protein 2 that associate with angiogenesis regulation and macrophage proliferation. THK cells were subcultured more than 90 times and can be useful for investigating the development and functioning of the teleostean innate immune system.

Giant seaperch iridovirus (GSIV) induces mitochondria-mediated cell death that is suppressed by bongkrekic acid and cycloheximide in a fish cell line.

Giant seaperch iridovirus (GSIV) induces cell death by an unknown mechanism. We postulated that this mechanism involves mitochondria-mediated cell death. Cell viability assays revealed a steady increase in dead grouper fin cells (GF-1) after GSIV infection, from 11% at 2 days post-infection (dpi) to 67% at 5 dpi. Annexin V/PI staining revealed GSIV infection induced apoptosis in a steadily increasing fraction of cells, from 4% at 1 dpi to 29% at 5 dpi. Furthermore, post-apoptotic necrosis was apparent at 4 and 5 dpi in the late replication stage. In the early replication stage, JC-1 dye revealed mitochondrial membrane potential (ΔΨm) loss in 42% of infected cells at 1 dpi, increasing to 98% at 3 dpi. Phosphatidylserine (PS) exposure and loss of ΔΨm from apoptosis/necrosis was attenuated by treatment with the adenine nucleotide translocase inhibitor bongkrekic acid (BKA) and the protein synthesis inhibitor cyclohexamide (CHX). These data suggest GSIV induces GF-1 apoptotic/necrotic cell death through pathways that require newly synthesized protein and involve the mitochondrial function.

Giant seaperch iridovirus infection upregulates Bas and Bak expression, leading to apoptotic death of fish cells.

The giant seaperch iridovirus (GSIV) induces host cell apoptosis by a poorly-understood process. In this study, GSIV is shown to upregulate the pro-apoptotic death genes Bax and Bak at the middle replication stage, and factors in the grouper fin cell line (GF-1) are shown to modulate this process. Studying the mechanism of cell death, we found that upregulated, de novo-synthesized Bax and Bak proteins formed heterodimers. This up-regulation process correlated with mitochondrial membrane potential (MMP) loss, increased caspase-3 activity, and increased apoptotic cell death. All effects were diminished by treatment of infected GF-1 cells with the protein synthesis inhibitor cycloheximide. Interestingly, overexpression of the anti-apoptotic gene Bcl-xL also diminished GSIV-induced mitochondria-mediated cell death, increasing host cell viability and decreasing MMP loss at the early replication stage. Our data suggest that GSIV induces GF-1 apoptotic cell death through up-regulation of the pro-apoptotic genes Bax and Bak, which are regulated by Bcl-xL overexpression on mitochondria in GF-1 cells.

Immunochemical and molecular characterization of a novel cell line derived from the brain of Trachinotus blochii (Teleostei, Perciformes): A fish cell line with oligodendrocyte progenitor cell and tanycyte characteristics.

Ependymal radial glial cells, also called tanycytes, are the predominant glial fibrillary acidic protein (GFAP)- and vimentin (VIM)-expressing cells in fish ependyma. Radial glial cells have been proposed to be neural stem cells but their molecular expression is not well understood. Previous studies revealed that fish neural progenitor and neural stem cells have A2B5, a marker for oligodendrocyte progenitor cells (OPCs). In this study, an A2B5(+) cell line, SPB, was isolated from the brain of the teleost Trachinotus blochii and characterized. SPB cells usually grew as polygonal epithelial cells, but at high density, long processes were commonly observed. Using immunocytochemistry, SPB cells were shown to exhibit oligodendrocyte markers such as galactocerebroside and Olig2, and radial glial cell markers such as brain lipid-binding protein, GFAP, Sox2, and VIM. SPB cells were also observed to have DARPP-32, a marker for tanycytes in mammals, and primary cilia. RT-PCR additionally revealed expression of bone morphogenetic protein 4, connexin35, Noggin2, and proteolipid protein in SPB cells. Results of this study suggest that SPB cells are OPCs that can display tanycyte characteristics. Fish tanycytes can be neural stem cells suggesting that SPB cells are neural stem cells. SPB is the first fish cell line showing primary cilia and markers for both OPCs and tanycytes.

Immunochemical and molecular characterization of GBC4 as a tanycyte-like cell line derived from grouper brain.

A clonal cell line, GBC4, derived from grouper (Epinephelus coioides) brain is proposed to represent an immature astroglial cell line because it expresses glial fibrillary acidic protein (GFAP), cytokeratin and vimentin. In teleost brain, tanycytes are the most abundant GFAP-expressing cell type, suggesting that GBC4 cells are derived from tanycytes. To test this hypothesis, protein and mRNA expression profiles of GBC4 cells were evaluated. We detected protein and/or mRNA expression of aromatase B, brain lipid binding protein, connexin43 protein, glutamine synthetase, S100 protein and Sox2. These proteins/mRNAs are also expressed in fish tanycytes. GBC4 cells also contained oligodendroglia proteins, including A2B5, galactocerebroside, myelin basic protein, proteolipid protein and platelet-derived growth factor receptor alpha as well as certain neuronal protein markers such as connexin35 protein and tyrosine hydroxylase. Our results indicate that GBC4 cells may be multipotent neural progenitor cells similar to tanycytes. Because GBC4 expresses several neural-specific genes, this line will be useful for studies on gene expression/regulation and neural development.

Isolation and characterization of a neural progenitor cell line from tilapia brain.

Astroglial cell lines have many applications for advancing neural developmental and functional studies. However, few astroglial cell lines have been reported from fish. In this study, we report the characterization of the immortal cell line TB2 isolated from adult tilapia brain tissue. The cell line was established at 25 degrees C in L15 medium supplemented with 15% fetal bovine serum. Most of the cells displayed a fibrous morphology and were immunoreactive for A2B5 antigen, glial fibrillary acidic protein (GFAP), keratin, vimentin, and the gap junction protein connexin 43 (Cx43). They weakly expressed glutamine synthetase (GS), S100 protein, and the neural stem cell markers Sox2 and brain lipid binding protein (BLBP). In contrast to astroglia in vivo, most TB2 cells also expressed galactocerebroside (GalC), substance P (SP), and tyrosine hydroxylase (TH). By immunoblot and RT-PCR, the cells also expressed myelin basic protein (MBP), proteolipid protein (PLP), and Cx35. On a poly-L-lysine-coated substrate in vitro, TB2 cells showed increases in neuronal dopamine decarboxylase (DDC) and microtubule-associated protein 2 (MAP2), indicating that they can initiate differentiation into neurons. Taken together, the results suggest that TB2 cells are astroglial progenitor cells (neural stem cells) and may develop into oligodendrocytes and neurons in a suitable environment. The present study advances our knowledge of fish astroglia. However, the factors that affect neural development in fish remain unknown, as do the characteristics of the intermediate differentiation stages between stem cells and mature nerve cells. The TB2 cell line will promote these investigations.