Dataset Information

Surface display of hirame novirhabdovirus (HIRRV) G protein in Lactococcus lactis and its immune protection in flounder (Paralichthys olivaceus).

ABSTRACT:

Background

Hirame novirhabdovirus (HIRRV) can infect a wide range of marine and freshwater fish, causing huge economic losses to aquaculture industry. Vaccine development, especially oral vaccine, has become an effective and convenient way to control aquatic infectious diseases. HIRRV glycoprotein (G), an immunogenic viral protein is a potential vaccine candidate for prevention of the disease. Here, we aimed to construct a recombinant Lactococcus lactis strain expressing HIRRV-G on the cell surface as an oral vaccine to prevent HIRRV.

Results

Glycoprotein gene of HIRRV was successfully cloned and expressed in L. lactis NZ9000 in a surface-displayed form, yielding Ll:pSLC-G. An approximately 81 kDa recombinant G protein (containing LysM anchoring motif) was confirmed by SDS-PAGE, western blotting and mass spectrometry analysis. The surface-displayed G protein was also verified by immunofluorescence and flow cytometry assays. Furthermore, to evaluate the potential of Ll:pSLC-G as oral vaccine candidate, flounders were continuously fed with commercial diet pellets coated with 1.0?×?10⁹ cfu/g of induced Ll:pSLC-G for 1 week. Four weeks later, booster vaccination was performed with the same procedure. Compared with the controls, Ll:pSLC-G elicited significantly higher levels of specific IgM against HIRRV in flounder gut mucus at the second week and in serum at the fourth week (p?4-10⁵ recombinant L. lactis cells were detected in every gram of foregut, midgut and hindgut of flounder, which were mainly localized at the bottom of gut mucus layer; and on day 21, 10²-10³ L. lactis cells could still be recovered.

Conclusions

HIRRV-G protein was successfully expressed on the surface of L. lactis cells, which could trigger mucosal and humoral immune response of flounder and provide considerable immune protection against HIRRV. It suggests that genetically engineered L. lactis expressing G protein can be employed as a promising oral vaccine against HIRRV infection.

SUBMITTER: Zhao L

PROVIDER: S-EPMC6704618 | biostudies-literature | 2019 Aug

REPOSITORIES: biostudies-literature

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Publications

Surface display of hirame novirhabdovirus (HIRRV) G protein in Lactococcus lactis and its immune protection in flounder (Paralichthys olivaceus).

Zhao Lining L Tang Xiaoqian X Sheng Xiuzhen X Xing Jing J Zhan Wenbin W

Microbial cell factories 20190821 1

<h4>Background</h4>Hirame novirhabdovirus (HIRRV) can infect a wide range of marine and freshwater fish, causing huge economic losses to aquaculture industry. Vaccine development, especially oral vaccine, has become an effective and convenient way to control aquatic infectious diseases. HIRRV glycoprotein (G), an immunogenic viral protein is a potential vaccine candidate for prevention of the disease. Here, we aimed to construct a recombinant Lactococcus lactis strain expressing HIRRV-G on the c ...[more]

PMID: 31434565

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Project description:Hirame novirhabdovirus (HIRRV) is one of most serious viral pathogens causing significant economic losses to the flounder (Paralichthys olivaceus)-farming industry. Previous studies have shown that the outbreak of HIRRV is highly temperature-dependent, and revealed the viral replication was significantly affected by the antiviral response of flounders under different temperatures. In the present study, the proteome and phosphoproteome was used to analyze the different antiviral responses in the HIRRV-infected flounder under 10 °C and 20 °C. Post viral infection, 472 differentially expressed proteins (DEPs) were identified in the spleen of flounder under 10 °C, which related to NOD-like receptor signaling pathway, RIG-I-like receptor signaling pathway, RNA transport and so on. Under 20 °C, 652 DEPs were identified and involved in focal adhesion, regulation of actin cytoskeleton, phagosome, NOD-like receptor signaling pathway and RIG-I-like receptor signaling pathway. Phosphoproteome analysis showed that 675 differentially expressed phosphoproteins (DEPPs) were identified in the viral infected spleen under 10 °C and significantly enriched in Spliceosome, signaling pathway, necroptosis and RNA transport. Under 20 °C, 1304 DEPPs were identified and significantly enriched to Proteasome, VEGF signaling pathway, apoptosis, Spliceosome, mTOR signaling pathway, mRNA surveillance pathway, and RNA transport. To be noted, the proteins and phosphoproteins involved in interferon production and signaling showed significant upregulations in the viral infected flounder under 20 °C compared with that under 10 °C. Furthermore, the temporal expression profiles of eight selected antiviral-related mRNA including IRF3, IRF7, IKKβ, TBK1, IFIT1, IFI44, MX1 and ISG15 were detected by qRT-PCR, which showed a significantly stronger response at early infection under 20 °C. These results provided fundamental resources for subsequent in-depth research on the HIRRV infection mechanism and the antiviral immunity of flounder, and also gives evidences for the high mortality of HIRRV-infected flounder under low temperature.

Project description:Hirame novirhabdovirus (HIRRV) is an ongoing threat to the aquaculture industry. The water temperature for the onset of HIRRV is below 15°C, the peak is about 10°C, but no mortality is observed over 20°C. Previous studies found the positive signal of matrix protein of HIRRV (HIRRV-M) was detected in the peripheral blood leukocytes of viral-infected flounder. Flow cytometry and indirect immunofluorescence assay showed that HIRRV-M was detected in mIgM+ B lymphocytes in viral-infected flounder maintained at 10°C and 20°C, and 22% mIgM+ B lymphocytes are infected at 10°C while 13% are infected at 20°C, indicating that HIRRV could invade into mIgM+ B lymphocytes. Absolute quantitative RT-PCR showed that the viral copies in mIgM+ B lymphocytes were significantly increased at 24 h post infection (hpi) both at 10°C and 20°C, but the viral copies in 10°C infection group were significantly higher than that in 20°C infection group at 72 hpi and 96 hpi. Furthermore, the B lymphocytes were sorted from HIRRV-infected flounder maintained at 10°C and 20°C for RNA-seq. The results showed that the differentially expression genes in mIgM+ B lymphocyte of healthy flounder at 10°C and 20°C were mainly enriched in metabolic pathways. Lipid metabolism and Amino acid metabolism were enhanced at 10°C, while Glucose metabolism was enhanced at 20°C. In contrast, HIRRV infection at 10°C induced the up-regulation of the Complement and coagulation cascades, FcγR-mediated phagocytosis, Platelets activation, Leukocyte transendothelial migration and Natural killer cell mediated cytotoxicity pathways at 72 hpi. HIRRV infection at 20°C induced the up-regulation of the Antigen processing and presentation pathway at 72 hpi. Subsequently, the temporal expression patterns of 16 genes involved in Antigen processing and presentation pathway were investigated by qRT-PCR, and results showed that the pathway was significantly activated by HIRRV infection at 20°C but inhibited at 10°C. In conclusion, HIRRV could invade into mIgM+ B lymphocytes and elicit differential immune response under 10°C and 20°C, which provide a deep insight into the antiviral response in mIgM+ B lymphocytes.

Dataset Information

Surface display of hirame novirhabdovirus (HIRRV) G protein in Lactococcus lactis and its immune protection in flounder (Paralichthys olivaceus).

Background

Results

Conclusions

Publications

Surface display of hirame novirhabdovirus (HIRRV) G protein in Lactococcus lactis and its immune protection in flounder (Paralichthys olivaceus).

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