Search

Infectious hematopoietic necrosis virus (IHNV) is an important pathogen that causes significant economic losses to salmon trout farming. Although vaccines have been invented for the treatment of IHNV, findings from our previous survey show that breeding enterprises and farmers require effective oral drugs or immune enhancers. However, studies on the development of oral drugs are limited. In the present study, we used bioinformatics methods to predict the protein targets of andrographolide (Andro) in IHNV. Cells were infected with IHNV, and the effect of andrographolide was explored by evaluating the expression levels of genes implicated in oxidative stress, activities of antioxidant enzymes, and the expression of genes implicated in apoptosis and necrosis. In the present study, cells were divided into NC, IHNV, IHNV+10 μM andrographolide, and IHNV+20 μM andrographolide groups. qRT-PCR was performed to determine the expression level of genes, and an antioxidant enzyme detection kit was used to evaluate the activities of antioxidant enzymes. Fluorescent staining was performed using a reactive oxygen species detection kit (ROS) and Hoechst 33342/PI double staining kit, and the mechanism of alleviation of apoptosis and oxidative stress andrographolide after IHNV infection was determined. The results indicated that andrographolide inhibits viral growth by binding to the NV protein of IHNV and increasing the antioxidant capacity of the body through the CTSK/BCL2/Cytc axis, thereby inhibiting the occurrence of IHNV-induced apoptosis. This is the first study to explore the antagonistic mechanism of action of andrographolide in alleviating IHNV infection. The results provide valuable information on alternative strategies for the treatment of IHNV infection during salmon family and provide a reference for the use of andrographolide as an antioxidant agent in agricultural settings. [ABSTRACT FROM AUTHOR]

MicroRNAs (miRNAs) are a class of regulators essential for numerous biological processes. Infectious hematopoietic necrosis virus (IHNV) is one of the most important viral pathogens in salmon and trout. In this study, the miRNA expression profiles of rainbow trout upon IHNV infection were explored. In total, 392 known miRNAs and 936 novel miRNAs were identified. Twelve known and 13 novel miRNAs were differentially expressed between infected and uninfected fish. Gene Ontology and Kyoto Encyclopedia of Genes and Genomes analyses revealed that certain miRNA target genes were associated with biological regulation, the immune system, and signal transduction. In addition, over- and suppressed expression of miR-146a-3p, miR-155–5p, miR-216a-5p, and miR-499b-5p could respectively increase and decrease viral gene expression in cells and viral titers. MiR-146a-3p and miR-216a-5p inhibited the expression of type-I IFN and the Mx1 gene induced by IHNV. These results provide preliminary insights into the IHNV–host interactions mediated by miRNAs. [ABSTRACT FROM AUTHOR]

Objective: To investigate the immunogenicity of different regions of glycoproteins in fish infectious hematopoietic necrosis virus (IHNV)., Methods: The glycoproteins was truncated and expressed according to the prediction of antigenicity and hydrophobicity by DNAStar 6.0 software. The protein was purified and used to produce antisera in rabbits. ELISA was performed to determine the antiserum titer and the immunogenicity of the protein was tested by indirect immunofluorescence assay (IFA)., Results: SDS-PAGE showed that protein with expected size 40 kDa was obtained. Purity of the obtained protein was as high as 90% as confirmed by high performance liquid chromatography (HPLC) analysis. The antisera against glycoprotein prepared in this study could react specifically with both natural glycoprotein of the IHNV-Sn1203 and the recombinant truncated glycoprotein in ELISA test, and the antisera titer against the natural glycoprotein and the recombinant glycoprotein was 1:20 000 and 1:80 000, respectively. IFA result showed that the antisera could recognize the glycoprotein on the surface of IHNV-Sn1203 and reference strain IHNV-WRAC., Conclusion: The antigenicity of the truncated glycoprotein produced in the study was as good as that of natural glycoprotein of IHNV.

The aim of the study is to establish a platform to deliver therapeutic proteins into target cells through a polyarginine-based cell penetrating peptide. To facilitate the expression of therapeutic proteins, a pSUMO (Small Ubiquitin-like Modifier)-R9-EGFP (enhanced green fluorescence protein) prokaryotic expression vector was constructed. After induction, the fusion protein SUMO-R9-EGFP was efficiently expressed. To validate the cell penetrating ability of the fusion protein, HepG2 cells were incubated with the purified R9-EGFP or EGFP protein as control, internalization of the fluorescent proteins was examined by either flow cytometry or confocal microscopy. The result obtained by flow cytometry showed that the R9-EGFP fusion protein could efficiently penetrate into the HepG2 cells in a dose and time-dependent manner. In contrast, the fluorescence was barely detected in the HepG2 cells incubated with EGFP control. The fluorescence intensity of the R9-EGFP treated cells reached plateau phase after 1.5 h. The result obtained by confocal microscopy shows that R9-EGFP efficiently entered into the HepG2 cells and was exclusively located in the cytoplasm, whereas, no fluorescence was detected in the cells incubated with the EGFP control. The heparin inhibition experiment showed that heparin could inhibit penetrating effect of the R9-EGFP protein by about 50%, suggesting that the penetrating ability of the fusion protein is heparin-dependent. In summary, the study has established a platform to deliver therapeutic proteins into target cells through a polyarginine-based penetrating peptide.

FGF-21 is a potential candidate for the treatment of type 2 diabetes mellitus. However, the clinical application of wild-type human FGF-21 is challenging due to some limitations, such as its poor hypoglycemic potency and short in vivo half-life. In this paper, we have produced an FGF-21 mutant (ahmFGF-21) by exchanging the functional domain of hFGF-21 with that of mFGF-21 to improve the potency of FGF-21. Results showed that the ahmFGF-21 protein was more potent than wild-type hFGF-21 in stimulating glucose uptake in vitro and lowering blood glucose levels of diabetic animals. To decrease its immunogenicity and increase its biostability, the N-terminus of ahmFGF-21 was modified in a sitespecific manner with 20 kDa mPEG-propionaldehyde (mPEG-ALD). We found that the preservation time of ahmFGF-21 in vitro was significantly prolonged after PEGylation. The serum antibody levels against ahmFGF-21 in immunized rabbits with the PEGylated ahmFGF-21 were significantly reduced than those with the unmodified ahmFGF-21, and the target protein concentration in the rabbits administrated with the PEGylated ahmFGF-21 increased 9.5-fold higher than that of the unmodified ahmFGF-21. The animal experimental results showed that PEGylation of ahmFGF-21 enhanced the hypoglycemic effect in diabetic mice. These results suggest that the in vitro and in vivo hypoglycemic effects of FGF-21 are significantly enhanced by genetic modification and the metabolic pharmacology of FGF-21 in type 2 diabetic mice is improved by PEGylation at a specific site.