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Construction of a recombinant eukaryotic vector for a grass carp reovirus VP6 gene and its expression in vitro and in vivo.

ABSTRACT: A recombinant plasmid expressing the VP6 inner capsid coding gene of grass carp reovirus (GCRV) was constructed and expressed in a Ctenopharyngodon idellus kidney (CIK) cell line and grass carps. The VP6 gene was amplified by RT-PCR, cloned into a pEGFP-N1 eukaryotic expression vector and transfected into CIK cells. Results from enhanced green fluorescent protein (EGFP) experiments and flow cytometry showed highest protein expression at 48 h. The immunoreactivity of fusion protein was confirmed using an indirect immunofluorescent assay. The specific binding between the fusion protein and polyclonal mouse GCRV VP6-specific antiserum indicated that the fusion protein was translated in vitro and had good immunogenicity. An antiviral activity assay showed that the virus titer was 100-fold lower in the GCRV VP6 expressed cells than in the pEGFP-N1 transfected cells. The expression levels of three immune genes in the head kidney of grass carps injected with the recombinant plasmid were used. Mx, TLR3 and IgM mRNA expression increased sharply at the 1st and 15th days post-injection (dpi). Specific antibodies were detected 30 days after vaccination. Neutralizing titers of the antibodies in vaccinated fish detected ranged from 160 to 320. Intramuscular injection of grass carps with 1 ?g of pEGFP-N1-VP6 was found to provide strong protection against GCRV. These results suggested that the VP6 gene was a good candidate for the design of GCRV-DNA vaccines and to investigate the use of cytokines as co-stimulatory molecules.

SUBMITTER: Zhou Y

PROVIDER: S-EPMC3889242 | biostudies-literature | 2014 Jan

REPOSITORIES: biostudies-literature

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Construction of a recombinant eukaryotic vector for a grass carp reovirus VP6 gene and its expression in vitro and in vivo.

Zhou Yong Y Fan Yu-Ding YD Zeng Ling-Bing LB

Virusdisease 20131130 1

A recombinant plasmid expressing the VP6 inner capsid coding gene of grass carp reovirus (GCRV) was constructed and expressed in a Ctenopharyngodon idellus kidney (CIK) cell line and grass carps. The VP6 gene was amplified by RT-PCR, cloned into a pEGFP-N1 eukaryotic expression vector and transfected into CIK cells. Results from enhanced green fluorescent protein (EGFP) experiments and flow cytometry showed highest protein expression at 48 h. The immunoreactivity of fusion protein was confirmed ...[more]

PMID: 24426312

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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.

Project description:BackgroundThe grass carp hemorrhagic disease caused by the grass carp reovirus (GCRV) is a major disease that hampers the development of grass carp aquaculture. The mechanism underlying GCRV pathogenesis and hemorrhagic symptoms is still unknown. MicroRNAs (miRNAs) are key regulators involved in various biological processes. The aim of this study was to identify conserved and novel miRNAs in grass carp in response to GCRV infection, as well as attempt to reveal the mechanism underlying GCRV pathogenesis and hemorrhagic symptoms.ResultsGrass carp were infected with GCRV, and spleen samples were collected at 0 (control), 1, 3, 5, 7, and 9 days post-infection (dpi). These samples were used to construct and sequence small RNA libraries. A total of 1208 miRNAs were identified, of which 278 were known miRNAs and 930 were novel miRNAs. Thirty-six miRNAs were identified to exhibit differential expression when compared with the control, and 536 target genes were predicted for the 36 miRNAs. GO and KEGG enrichment analyses of these target genes showed that many of the significantly enriched terms were associated with immune response, blood coagulation, hemostasis, and complement and coagulation cascades, especially the GO term "blood coagulation" and pathway "complement and coagulation cascades." Ten representative target genes involved in "complement and coagulation cascades" were selected for qPCR analysis, and the results showed that the expression patterns of these target genes were significantly upregulated at 7 dpi, suggesting that the pathway "complement and coagulation cascades" was strongly activated.ConclusionConserved and novel miRNAs in response to GCRV infection were identified in grass carp, of which 278 were known miRNAs and 930 were novel miRNAs. Many of the target genes involved in immune response, blood coagulation, hemostasis, and complement and coagulation cascades. Strong activation of the pathway "complement and coagulation cascades" may have led to endothelial-cell and blood-cell damage and hemorrhagic symptoms. The present study provides a new insight into understanding the mechanism underlying GCRV pathogenesis and hemorrhagic symptoms.

Dataset Information

Construction of a recombinant eukaryotic vector for a grass carp reovirus VP6 gene and its expression in vitro and in vivo.

Publications

Construction of a recombinant eukaryotic vector for a grass carp reovirus VP6 gene and its expression in vitro and in vivo.

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