Characterization and antiviral function of a cytosolic sensor gene, MDA5, in Japanese flounder, Paralichthys olivaceus.
ABSTRACT Cytosolic pattern recognition receptors such as retinoic acid-inducible gene I (RIG-I)-like receptors (RLRs) play an important role in sensing viral RNAs. The receptor encoded by melanoma differentiation-associated gene 5 (MDA5), an RLR, recognizes viral RNA in the cytoplasm and enhances antiviral response in host cells. The full-length MDA5 gene in Japanese flounder, Paralichthys olivaceus was cloned and found to have 11,251 nucleotides. MDA5 transcript abundance was significantly increased in whole kidney infected with viral hemorrhagic septicemia virus (VHSV) as well as whole kidney and peripheral blood leukocytes stimulated with poly I:C in vitro. Hirame natural embryo (HINAE) cells overexpressing MDA5 showed a lower cytopathic effect (CPE) against VHSV, hirame rhabdovirus (HIRRV) and infectious pancreatic necrosis virus (IPNV) infection. When infected with VHSV, MDA5-overexpressing HINAE cells had 24-75 fold lower virus titer than normal HINAE cells. These results suggest that Japanese flounder MDA5 is involved in the induction of antiviral response.
Article: Both STING and MAVS Fish Orthologs Contribute to the Induction of Interferon Mediated by RIG-I.[show abstract] [hide abstract]
ABSTRACT: Viral infections are detected in most cases by the host innate immune system through pattern-recognition receptors (PRR), the sensors for pathogen-associated molecular patterns (PAMPs), which induce the production of cytokines, such as type I interferons (IFN). Recent identification in mammalian and teleost fish of cytoplasmic viral RNA sensors, RIG-I-like receptors (RLRs), and their mitochondrial adaptor: the mitochondrial antiviral signaling (MAVS) protein, also called IPS-1, highlight their important role in the induction of IFN at the early stage of a virus infection. More recently, an endoplasmic reticulum (ER) adaptor: the stimulator of interferon genes (STING) protein, also called MITA, ERIS and MPYS, has been shown to play a pivotal role in response to both non-self-cytosolic RNA and dsDNA. In this study, we cloned STING cDNAs from zebrafish and showed that it was an ortholog to mammalian STING. We demonstrated that overexpression of this ER protein in fish cells led to a constitutive induction of IFN and interferon-stimulated genes (ISGs). STING-overexpressing cells were almost fully protected against RNA virus infection with a strong inhibition of both DNA and RNA virus replication. In addition, we found that together with MAVS, STING was an important player in the RIG-I IFN-inducing pathway. This report provides the demonstration that teleost fish possess a functional RLR pathway in which MAVS and STING are downstream signaling molecules of RIG-I. The Sequences presented in this article have been submitted to GenBank under accession numbers: Zebrafish STING (HE856619); EPC STING (HE856620); EPC IRF3 (HE856621); EPC IFN promoter (HE856618).PLoS ONE 01/2012; 7(10):e47737. · 4.09 Impact Factor
Article: Impaired TLR2 and TLR7 response in olive flounder infected with viral haemorrhagic septicaemia virus at host susceptible 15°C but high at non-susceptible 20°C.[show abstract] [hide abstract]
ABSTRACT: The olive flounder (Paralichthys olivaceus) is susceptible to viral haemorrhagic septicaemia virus (VHSV) at 15°C but no mortality is observed at 20°C even though the virus can grow profusely in vitro. Thus, we designed an experiment to better understand the immune response of olive flounder to VHSV when the host reared at 15°C or 20°C and infected with the virus. Olive flounder (18-22g) reared at 15±0.5°C or 20±0.5°C were intra-peritoneally injected with VHSV (107.8 TCID50/fish) and sampled (n=5) for head kidney at 3, 6, 12 hpi, 1, 2, 4 and 7 dpi; similarly, mock injected control groups (n=5). Real-time PCR based absolute quantification method was followed to quantify copies of VHSV gRNA and mRNA, while the immune gene expression of the olive flounder was quantified relative to internal control, β-actin. Viral infection resulted in a cumulative mortality of 24% in olive flounder reared at 15°C, but no mortality was recorded in the 20°C group or control groups. TLR2 and TLR7 expression at 15°C was enhanced during early infection phase (3-6 hpi) and recovery phase (4-7 dpi) when viral transcription was low, but expression was significantly reduced (12 hpi-1 dpi) at peak infection period. However, the 20°C group showed low viral transcription and expressed high level of TLR7 and a moderately higher unchanged level of TLR2. In both the groups, TLR3 expression was unaffected. Nevertheless, expression of MDA5 and LGP2 increased significantly irrespective of rearing temperature at the time of peak infection, hence at 15°C VHSV down-regulated expression of TLR2 and TLR7 but not MDA5 or LGP2. Comparatively, at 15°C IRF3 expressed high but IRF7 remained very low. Interleukins (IL-1β, IL-6 and IL-8) were significantly elevated in both the groups, but quicker and for a shorter period at 20°C. In the 15°C group, an extended period of expression of ILs could create an unsafe prolonged inflammatory condition. The olive flounders expressed high ISGs at 15°C but were lagging by 12 hours than 20°C group. Based on these findings, we concluded that viral-mediated disruption of TLR2 and TLR7 expression in the 15°C group could have delayed the host interferon response and provided a window for high viral growth. However, an effective host immune response at 20°C contained VHSV from reaching the critical limit.Fish & Shellfish Immunology 02/2013; · 3.32 Impact Factor