Generation of viperin-knockout zebrafish by CRISPR/Cas9-mediated genome engineering and the effect of this mutation under VHSV infection.

Viperin is an important virus-induced protein in animals that negatively participates in RNA viral replication and transcription. The reactive machinery of viperin suggests that it produces a regulatory molecule ddhCTP, which may affect immune regulation. In this study, we investigated the expression pattern of viperin in larval and adult stages of zebrafish by whole-mount in situ hybridization and reverse transcription-quantitative PCR (RT-qPCR). To elucidate the function of viperin, we generated a zebrafish knockout model using the CRISPR/Cas9 method and evaluated the mutation's effects under viral hemorrhagic septicemia virus (VHSV) infections. In zebrafish larvae, viperin was expressed in the brain region, eye, and pharynx, which was confirmed by cryosectioning. In adult zebrafish, blood cells showed the highest levels of viperin expression. In 5 dpf fish challenged with VHSV, the expression of the viral NP protein was significantly enhanced in viperin-/- compared to wild-type fish. In vitro VHSV propagation analysis indicated comparatively higher levels of virus propagation in viperin-/- fish. Mortality analysis confirmed higher mortality rates, and interferon gene expression analysis showed a strong upregulation of interferon (ifn)φ1 and 3 gene in viperin-/- fish infected with VHSV. This study describes the successful generation of a viperin-knockout model and the role of viperin during VHSV infections.

A copper-zinc-superoxide dismutase (CuZnSOD) from redlip mullet, Liza haematocheila: Insights to its structural characteristics, immune responses, antioxidant activity, and potent antibacterial properties.

Copper-zinc superoxide dismutase (CuZnSOD) is a nuclear-encoded metalloenzyme responsible for scavenging harmful reactive oxygen species (ROS). In this study, the CuZnSOD homolog from redlip mullet (Liza haematochelia) (MuCuZnSOD) was structurally and functionally characterized to evaluate its antioxidant capacity, antibacterial properties, and protective level in various pathogenic stress conditions. Structural characteristics of MuCuZnSOD were evaluated using different bioinformatics tools. Pairwise sequence comparison and evolutionary tree structure revealed that the MuCuZnSOD sequence was closely related to the CuZnSOD sequence of Oplegnathus fasciatus with a 94.2% sequence identity. Sequence alignment analysis indicated that the CuZnSOD domain was well conserved. The highest transcriptional expression of MuCuZnSOD was identified in the blood. Immune challenge with lipopolysaccharide (LPS), Lactococcus garvieae, and polyinosinic-polycytidylic acid (poly I:C) exhibited an increased MuCuZnSOD mRNA expression in the blood and liver. Transfected green fluorescent protein-fused MuCuZnSOD was localized in the cytoplasm. Recombinant MuCuZnSOD (rMuCuZnSOD) was overexpressed in a bacterial system. The rMuCuZnSOD possessed significant antioxidant properties as determined by conventional xanthine oxidase assay. The optimum pH and temperature of rMuCuZnSOD were found to be pH 9 and 25 °C, respectively. rMuCuZnSOD enzyme activity increased in a concentration-dependent manner. Treatment with potassium cyanide highly inhibited the rMuCuZnSOD activity. rMuCuZnSOD possessed a significant peroxidation activity in the presence of HCO3- ions as demonstrated by the increased viability in cells treated with the enzyme in the presence of hydrogen peroxide. Antibacterial assays showed that rMuCuZnSOD had significant growth-inhibitory effects on both gram-positive and gram-negative bacteria. Collectively, our findings demonstrate that MuCuZnSOD is an essential antioxidant protein, which regulates the host defense mechanisms and innate immunity under oxidative stress.

Molecular profiling and functional delineation of peroxiredoxin 3 (HaPrx3) from the big-belly seahorses (Hippocampus abdominalis) and understanding their immunological responses.

Peroxiredoxins (Prxs) are ubiquitously expressed antioxidant proteins that can protect aerobic organisms from oxidative stress. Here, we characterized the HaPrx3 homolog at the molecular level from big-belly seahorse (Hippocampus abdominalis) and analyzed its functional activities. The coding sequence of HaPrx3 consists of 726 bp, which encodes 241 amino acids. The predicted molecular weight and theoretical isoelectric point (pI) of HaPrx3 was 26.20 kDa and 7.04, respectively. Multiple sequence alignments revealed that the arrangements of domains, catalytic triads, dimers, and decamer interfaces of HaPrx3 were conserved among Prx sequences of other organisms. According to the phylogenetic analysis, HaPrx3 is clustered with the teleost Prx3 subclade. The highest transcript level of HaPrx3 was detected in the ovary tissue among fourteen healthy fish tissues. The mRNA levels of HaPrx3 in blood and liver tissues were significantly (P < 0.05) upregulated in response to lipopolysaccharide (LPS), polyinosinic-polycytidylic (poly I:C), Edwardsiella tarda, and Streptococcus iniae, suggesting its involvement in immune responses. Under functional properties, insulin disulfide reduction assay confirmed the oxidoreductase activity of recombinant HaPrx3. A cell viability assay and Hoechst staining indicated cell survival ability and reduction of apoptotic activity, respectively. Moreover, a peroxidase activity assay verified peroxidase activity, while a metal-catalyzed oxidation (MCO) assay indicated the DNA protection ability of HaPrx3. Collectively, it is concluded that HaPrx3 may play a significant role in oxidative stress and immune responses against pathogenic infections in big-belly seahorses.

Molecular characterization and expression analysis of big-belly seahorse (Hippocampus abdominalis) interleukin-10 and analysis of its potent anti-inflammatory properties in LPS-induced murine macrophage RAW 264.7 cells.

Interleukin-10 (IL-10) is a pleiotropic cytokine involved in the regulation of innate and adaptive immunity. In this study, IL-10 from big-belly seahorse (Hippocampus abdominalis) (HaIL-10) was characterized based on its molecular and functional aspects. The coding sequence of HaIL-10 is 570 bp in length and encodes a 189-amino acid residue protein (calculated molecular weight, 21.89 kDa). The deduced amino acid sequence comprises a typical signal peptide and a mature peptide domain sequence carrying four conserved Cys residues and two additional Cys residues specific to fish. Phylogenetic analysis indicated an evolutionary relationship between HaIL-10 and its counterparts in other vertebrates, with close clustering to the fish-specific homologs. Recombinant HaIL-10 (rHaIL-10) significantly reduced nitric oxide (NO) production by lipopolysaccharide (LPS)-induced murine macrophage RAW 264.7 cells in a concentration-dependent manner but had no effect on cell viability, suggestive of its involvement in immune response. The protein expressions of iNOS and COX-2 were significantly reduced by rHaIL-10 in LPS-induced murine macrophages RAW 264.7 cells. HaIL-10 mRNA expression was observed in all analyzed tissues, with the maximum expression being noted in the kidney and ovary. However, transcriptional levels of HaIL-10 were significantly higher in the blood, gill, and intestine upon in vivo induction with LPS, polyinosinic:polycytidylic acid [poly (I:C)], and Streptococcus iniae. To summarize, our findings help in the improved understanding of the biological functions of HaIL-10 and modulation of HaIL-10 mRNA expression in response to immune stress.