Dataset Information

Functional characterizations of RIG-I to GCRV and viral/bacterial PAMPs in grass carp Ctenopharyngodon idella.

ABSTRACT:

Background

RIG-I (retinoic acid inducible gene-I) is one of the key cytosolic pattern recognition receptors (PRRs) for detecting nucleotide pathogen associated molecular patterns (PAMPs) and mediating the induction of type I interferon and inflammatory cytokines in innate immune response. Though the mechanism is well characterized in mammals, the study of the accurate function of RIG-I in teleosts is still in its infancy.

Methodology/principal findings

To clarify the functional characterizations of RIG-I in grass carp Ctenopharyngodon idella (CiRIG-I), six representative overexpression plasmids were constructed and transfected into C. idella kidney (CIK) cell lines to obtain stably expressing recombinant proteins, respectively. A virus titer test and 96-well plate staining assay showed that all constructs exhibited the antiviral activity somewhat. The quantitative real-time RT-PCR (qRT-PCR) demonstrated that mRNA expressions of CiIPS-1, CiIFN-I and CiMx2 were regulated by not only virus (GCRV) or viral PAMP (poly(IC)) challenge but also bacterial PAMPs (LPS and PGN) stimulation in the steadily transfected cells. The results showed that the full-length CiRIG-I played a key role in RLR pathway. The repressor domain (RD) exerted an inhibitory function of the signaling channel under all utilized challenges. Caspase activation and recruitment domains (CARDs) showed a positive role in GCRV and poly(I:C) challenge. Helicase motifs were crucial for the signaling pathway upon LPS and PGN stimulation. Interestingly, ?CARDs (CARDs deleted) showed positive modulation in RIG-I signal transduction.

Conclusions/significance

The results provided some novel insights into RIG-I sensing with a strikingly broad regulation in teleosts, responding not only to the dsRNA virus or synthetic dsRNA but also bacterial PAMPs.

SUBMITTER: Chen L

PROVIDER: S-EPMC3409128 | biostudies-literature | 2012

REPOSITORIES: biostudies-literature

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Publications

Functional characterizations of RIG-I to GCRV and viral/bacterial PAMPs in grass carp Ctenopharyngodon idella.

Chen Lijun L Su Jianguo J Su Jianguo J Yang Chunrong C Peng Limin L Wan Quanyuan Q Wang Lan L

PloS one 20120731 7

<h4>Background</h4>RIG-I (retinoic acid inducible gene-I) is one of the key cytosolic pattern recognition receptors (PRRs) for detecting nucleotide pathogen associated molecular patterns (PAMPs) and mediating the induction of type I interferon and inflammatory cytokines in innate immune response. Though the mechanism is well characterized in mammals, the study of the accurate function of RIG-I in teleosts is still in its infancy.<h4>Methodology/principal findings</h4>To clarify the functional ch ...[more]

PMID: 22860079

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Project description:The grass carp reovirus (GCRV) causes severe hemorrhagic disease with high mortality and leads to serious economic losses in the grass carp (Ctenopharyngodon idella) industry in China. Oral vaccine has been proven to be an effective method to provide protection against fish viruses. In this study, a recombinant baculovirus BmNPV-VP35-VP4 was generated to express VP35 and VP4 proteins from GCRV type ? via Bac-to-Bac baculovirus expression system. The expression of recombinant VP35-VP4 protein (rVP35-VP4) in Bombyx mori embryo cells (BmE) and silkworm pupae was confirmed by Western blotting and immunofluorescence assay (IFA) after infection with BmNPV-VP35-VP4. To vaccinate the grass carp by oral route, the silkworm pupae expressing the rVP35-VP4 proteins were converted into a powder after freeze-drying, added to artificial feed at 5% and fed to grass carp (18 ± 1.5 g) for six weeks, and the immune response and protective efficacy in grass carp after oral vaccination trial was thoroughly investigated. This included blood cell counting and classification, serum antibody titer detection, immune-related gene expression and the relative percent survival rate in immunized grass carp. The results of blood cell counts show that the number of white blood cells in the peripheral blood of immunized grass carp increased significantly from 14 to 28 days post-immunization (dpi). The differential leukocyte count of neutrophils and monocytes were significantly higher than those in the control group at 14 dpi. Additionally, the number of lymphocytes increased significantly and reached a peak at 28 dpi. The serum antibody levels were significantly increased at Day 14 and continued until 42 days post-vaccination. The mRNA expression levels of immune-related genes (IFN-1, TLR22, IL-1?, MHC I, Mx and IgM) were significantly upregulated in liver, spleen, kidney and hindgut after immunization. Four weeks post-immunization, fish were challenged with virulent GCRV by intraperitoneal injection. The results of this challenge study show that orally immunized group exhibited a survival rate of 60% and relative percent survival (RPS) of 56%, whereas the control group had a survival rate of 13% and RPS of 4%. Taken together, our results demonstrate that the silkworm pupae powder containing baculovirus-expressed VP35-VP4 proteins could induce both non-specific and specific immune responses and protect grass carp against GCRV infection, suggesting it could be used as an oral vaccine.

Project description:BackgroundMyxovirus resistance (Mx) proteins are crucial effectors of the innate antiviral response against a wide range of viruses, mediated by the type I interferon (IFN-I) signaling pathway. However, the antiviral activity of Mx proteins is diverse and complicated in different species.Methodology/principal findingsIn the current study, two novel Mx genes (CiMx1 and CiMx3) were identified in grass carp (Ctenopharyngodon idella). CiMx1 and CiMx3 proteins exhibit high sequence identity (92.1%), and low identity with CiMx2 (49.2% and 49.5%, respectively) from the GenBank database. The predicted three-dimensional (3D) structures are distinct among the three isoforms. mRNA instability motifs also display significant differences in the three genes. The spatial and temporal expression profiles of three C. idella Mx genes and the IFN-I gene were investigated by real-time fluorescence quantitative RT-PCR (qRT-PCR) following infection with grass carp reovirus (GCRV) in vivo and in vitro. The results demonstrated that all the four genes were implicated in the anti-GCRV immune response, that mRNA expression of Mx genes might be independent of IFN-I, and that CIK cells are suitable for antiviral studies. By comparing expression patterns following GCRV challenge or poly(I:C) treatment, it was observed that GCRV blocks mRNA expression of the four genes. To determine the functions of Mx genes, three CiMx cDNAs were cloned into expression vectors and utilized for transfection of CIK cells. The protection conferred by each recombinant CiMx protein against GCRV infection was evaluated. Antiviral activity against GCRV was demonstrated by reduced cytopathic effect, lower virus titer and lower levels of expressed viral transcripts. The transcription of IFN-I gene was also monitored.Conclusions/significanceThe results indicate all three Mx genes can suppress replication of grass carp reovirus and over-expression of Mx genes mediate feedback inhibition of the IFN-I gene.

Dataset Information

Functional characterizations of RIG-I to GCRV and viral/bacterial PAMPs in grass carp Ctenopharyngodon idella.

Background

Methodology/principal findings

Conclusions/significance

Publications

Functional characterizations of RIG-I to GCRV and viral/bacterial PAMPs in grass carp Ctenopharyngodon idella.

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