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Bid-deficient fish delay grass carp reovirus (GCRV) replication and attenuate GCRV-triggered apoptosis.

ABSTRACT: Bid, BH3-interacting domain death agonist, is a pro-apoptotic BH3-only member of Bcl-2 family, playing an important role in apoptosis. In the study, Bid genes from grass carp (Ctenopharyngodon idellus) and rare minnow (Gobiocypris rarus), named CiBid and GrBid, were cloned and analyzed. Bid was constitutively expressed in all examined tissues of grass carp, but the expression level varied in different tissues. Following grass carp reovirus (GCRV) stimulation in vivo, Bid and apoptosis related genes Caspase-9 and Caspase-3 was up-regulated significantly at the late stage of infection. Moreover, we generated a Bid-deficient rare minnow (Bid-/-) to investigate the possible role of Bid in GCRV-triggered apoptosis. We found that the survival time of Bid-/- rare minnow after GCRV infection was extended when compared with wild-type fish, the relative copy number of GCRV in Bid-/- rare minnow was lower than that in wild-type fish, and the expression level of Caspase-9 and Caspase-3 in Bid-/- rare minnow were significantly lower than that in the wild-type fish. Collectively, the current data revealed the important role of Bid during virus-induced apoptosis in teleost fish. Our study would provide new insight into understanding the GCRV induced apoptosis and may provide a target gene for virus-resistant breeding in grass carp.

SUBMITTER: He L

PROVIDER: S-EPMC5652715 | biostudies-literature | 2017 Sep

REPOSITORIES: biostudies-literature

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Bid-deficient fish delay grass carp reovirus (GCRV) replication and attenuate GCRV-triggered apoptosis.

He Libo L Wang Hao H Luo Lifei L Li Yongming Y Huang Rong R Liao Lanjie L Zhu Zuoyan Z Wang Yaping Y

Oncotarget 20170722 44

Bid, BH3-interacting domain death agonist, is a pro-apoptotic BH3-only member of Bcl-2 family, playing an important role in apoptosis. In the study, Bid genes from grass carp (Ctenopharyngodon idellus) and rare minnow (Gobiocypris rarus), named CiBid and GrBid, were cloned and analyzed. Bid was constitutively expressed in all examined tissues of grass carp, but the expression level varied in different tissues. Following grass carp reovirus (GCRV) stimula ...[more]

PMID: 29100321

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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:Grass carp is an important farmed fish in China that is affected by serious disease, especially hemorrhagic disease caused by grass carp reovirus (GCRV). The mechanism underlying the hemorrhagic symptoms in infected fish remains to be elucidated. Although GCRV can be divided into three distinct subtypes, differences in the pathogenesis and host immune responses to the different subtypes are still unclear. The aim of this study was to provide a comprehensive insight into the grass carp response to different GCRV subtypes and to elucidate the mechanism underlying the hemorrhagic symptoms.Following infection of grass carp, GCRV-I was associated with a long latent period and low mortality (42.5%), while GCRV-II was associated with a short latent period and high mortality (81.4%). The relative copy number of GCRV-I remained consistent or decreased slightly throughout the first 7 days post-infection, whereas a marked increase in GCRV-II high copy number was detected at 5 days post-infection. Transcriptome sequencing revealed 211 differentially expressed genes (DEGs) in Group I (66 up-regulated, 145 down-regulated) and 670 (386 up-regulated, 284 down-regulated) in Group II. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis showed significant enrichment in the terms or pathways involved in immune responses and correlating with blood or platelets. Most of the DEGs in Group I were also present in Group II, although the expression profiles differed, with most DEGs showing mild changes in Group I, while marked changes were observed in Group II, especially the interferon-related genes. Many of the genes involved in the complement pathway and coagulation cascades were significantly up-regulated at 7 days post-infection in Group II, suggesting activation of these pathways.GCRV-I is associated with low virulence and a long latent period prior to the induction of a mild host immune response, whereas GCRV-II is associated with high virulence, a short latent period and stimulates a strong and extensive host immune response. The complement and coagulation pathways are significantly activated at 7 days post-infection, leading to the endothelial cell and blood cell damage that result in hemorrhagic symptoms.

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Bid-deficient fish delay grass carp reovirus (GCRV) replication and attenuate GCRV-triggered apoptosis.

Publications

<i>Bid</i>-deficient fish delay grass carp reovirus (GCRV) replication and attenuate GCRV-triggered apoptosis.

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