Characterization of viral entry and infection of quantum dot-labeled grass carp reovirus.
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ABSTRACT:
SUBMITTER: Zhang F
PROVIDER: S-EPMC6598975 | biostudies-literature | 2017 Apr
REPOSITORIES: biostudies-literature
Publications
Characterization of viral entry and infection of quantum dot-labeled grass carp reovirus.
Zhang Fuxian F Yan Shicui S Guo Hong H Chen Qingxiu Q Fang Qin Q
Virologica Sinica 20170401 2
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Project description:BACKGROUND: Grass Carp Reovirus (GCRV), a tentative member in the genus Aquareovirus of family Reoviridae, contains eleven segmented (double-stranded RNA) dsRNA genome which encodes 12 proteins. A low-copy core component protein VP4, encoded by the viral genome segment 5(S5), has been suggested to play a key role in viral genome transcription and replication. RESULTS: To understand the role of minor core protein VP4 played in molecular pathogenesis during GCRV infection, the recombinant GCRV VP4 gene was constructed and expressed in both prokaryotic and mammalian cells in this investigation. The recombinant His-tag fusion VP4 products expressed in E.coli were identified by Western blotting utilizing His-tag specific monoclonal and GCRV polyclonal antibodies. In addition, the expression of VP4 in GCRV infected cells, appeared in granules structure concentrated mainly in the cytoplasm, can be detected by Immunofluorescence (IF) using prepared anti-VP4 polyclonal antibody. Meanwhile, VP4 protein in GCRV core and infected cell lysate was identified by Immunoblotting (IB) assay. Of particular note, the VP4 protein was exhibited a diffuse distribution in the cytoplasm and nucleus in transfected cells, suggesting that VP4 protein may play a partial role in the nucleus by regulating cell cycle besides its predicted cytoplasmic function in GCRV infection. CONCLUSIONS: Our results indicate the VP4 is a core component in GCRV. The cellular localization of VP4 is correlated with its predicted function. The data provide a foundation for further studies aimed at understanding the role of VP4 in viroplasmic inclusion bodies (VIB) formation during GCRV replication and assembly.
| S-EPMC3390282 | biostudies-literature
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.
| S-EPMC5319172 | biostudies-literature
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.
| S-EPMC3522624 | biostudies-literature
Project description:Integrin β-1 (ITGB1) is a transmembrane protein belonging to the integrin family and it plays an important role in viral entry. In this study, the itgb1b gene of the rare minnow, Gobiocypris rarus, was cloned and analyzed. To investigate the possible role of itgb1b on grass carp reovirus (GCRV) infection, we generated an ITGB1b-deficient rare minnow (ITGB1b-/-) using the CRISPR/Cas9 system. Following stimulation with GCRV, the survival time of the -ITGB1b-/- rare minnows was extended in comparison to the wild-type minnows. Moreover, the relative copy number of GCRV and the level of clathrin-mediated endocytosis-associated and apoptosis-related gene expression in the ITGB1b-/- rare minnows was significantly lower than that of the wild-type minnows. These results suggested that the absence of itgb1b reduced viral entry efficiency and the expression of apoptosis-related genes. Moreover, the data suggested that itgb1b played an important role in mediating the entry of viruses into the cells via clathrin. Therefore, these findings provide novel insight into the function of itgb1b in the process of GCRV infection.
| S-EPMC6214113 | biostudies-literature
Project description:Viral inclusion bodies (VIBs) are subcellular structures required for efficient viral replication. How type II grass carp reovirus (GCRV-II), the mainly prevalent strain, forms VIBs is unknown. In this study, we found that GCRV-II infection induced punctate VIBs in grass carp ovary (GCO) cells and that non-structural protein 38 (NS38) functioned as a participant in VIB formation. Furthermore, VP56 and VP35 induced VIBs and recruited other viral proteins via the N-terminal of VP56 and the middle domain of VP35. Additionally, we found that the newly synthesized viral RNAs co-localized with VP56 and VP35 in VIBs during infection. Taken together, VP56 and VP35 induce VIB formation and recruit other viral proteins and viral RNAs to the VIBs for viral replication, which helps identify new targets for developing anti-GCRV-II drugs to disrupt viral replication.
| S-EPMC10767200 | biostudies-literature
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 Ⅱ 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-1β, 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.
| S-EPMC7827918 | biostudies-literature
Project description:RNA-seq of Ctenopharyngodon idellus kidney cells after GCRV infected 4hours and 8 hours.
2024-05-11 | E-MTAB-13002 | biostudies-arrayexpress
Project description:MeDIP-seq of Ctenopharyngodon idellus kidney cells after GCRV infected 8 hours.
2024-05-11 | E-MTAB-13003 | biostudies-arrayexpress
Project description:Global fish production from aquaculture has rapidly grown over the past decades, and grass carp shares the largest portion. However, hemorrhagic disease caused by grass carp reovirus (GCRV) results in tremendous loss of grass carp (Ctenopharyngodon idella) industry. During the past years, development of molecular biology and cellular biology technologies has promoted significant advances in the understanding of the pathogen and the immune system. Immunoprophylaxis based on stimulation of the immune system of fish has also got some achievements. In this review, authors summarize the recent progresses in basic researches on GCRV; viral nucleic acid sensors, high-mobility group box proteins (HMGBs); pattern recognition receptors (PRRs), Toll-like receptors (TLRs) and retinoic acid inducible gene I- (RIG-I-) like receptors (RLRs); antiviral immune responses induced by PRRs-mediated signaling cascades of type I interferon (IFN-I) and IFN-stimulated genes (ISGs) activation. The present review also notices the potential applications of molecule genetic markers. Additionally, authors discuss the current preventive and therapeutic strategies (vaccines, RNAi, and prevention medicine) and highlight the importance of innate immunity in long term control for grass carp hemorrhagic disease.
| S-EPMC4337036 | biostudies-literature
Project description:Grass carp is an important aquaculture fish species in China that is affected by severe diseases, especially haemorrhagic disease caused by grass carp reovirus (GCRV). However, the mechanisms of GCRV invasion and infection remain to be elucidated. In the present study, Ctenopharyngodon idellus kidney (CIK) cells were infected with GCRV, harvested at 0, 8, 24, and 72 h post infection, respectively, and then subjected to transcriptomics sequencing. Each sample yielded more than 6 Gb of clean data and 40 million clean reads. To better understand GCRV infection, the process was divided into three phases: the early (0-8 h post infection), middle (8-24 h post infection), and late (24-72 h) stages of infection. A total of 76 (35 up-regulated, 41 down-regulated), 553 (463 up-regulated, 90 down-regulated), and 284 (150 up-regulated, 134 down-regulated) differently expressed genes (DEGs) were identified during the early, middle, and late stages of infection, respectively. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis revealed that DEGs were mainly involved in carbohydrate biosynthesis, transport, and endocytosis in the early stage, phagocytosis and lysosome pathways were mainly enriched in the middle stage, and programmed cell death, apoptosis, and inflammation were largely associated with the late stage. These results suggest GCRV infection is a gradual process involving adsorption on the cell surface, followed by endocytosis into cells, transport by lysosomes, and eventually resulted in cell necrosis and/or apoptosis. Our findings provide insight into the mechanisms of grass carp reovirus infection.
| S-EPMC5855710 | biostudies-literature