Functional characterization of BAG3 in orange-spotted grouper (Epinephelus coioides) during viral infection.

Bcl-2-associated athanogene 3 (BAG3) is a cochaperone protein that interacts with Bcl-2 and mediate cell death. However, little is known about the roles of fish BAG3 during viral infection. In this study, we characterized a BAG3 homolog from orange-spotted grouper (Epinephelus coioides) (EcBAG3) and investigated its roles during viral infection. The EcBAG3 protein encoded 579 amino acids with typical WW, PXXP and BAG domains, which shared high identities with reported fish BAG3. Quantitative real-time PCR (qRT-PCR) analysis revealed that EcBAG3 was highly expressed in brain and heart. And the expression of EcBAG3 was significantly up-regulated after red-spotted grouper nervous necrosis virus (RGNNV) stimulation in vitro. EcBAG3 overexpression could promoted the expression of viral genes (coat protein (CP) and RNA-dependent RNA polymerase (RdRp)), which was enhanced by co-transfection with Hsp70 and Hsp22. Also, EcBAG3 overexpression up-regulated the expression of LC3-Ⅱ and down-regulated the expression of Bax and BNIP3, the IFN- (IRF1, IRF3, IRF7, IFP35, Mx1) or inflammation-related (IL-1ß and TNFα) factors, as well as decreased the activities of NF-κB, ISRE and IFN-3. While knockdown of EcBAG3 decreased the transcripts of RGNNV CP gene and RdRp gene. Further studies showed that EcBAG3 knockdown impaired the expression level of autophagy factor LC3-Ⅱ, and promoted the expression level of Bax and BNIP3, inflammatory factors and interferon factors. These data indicate that EcBAG3 can affect viral infection through modulating virus-induced cell death, regulating the expression of IFN- and inflammation-related factors, which will be helpful to further explore the immune response of fish during viral infection.

Identification of BAG5 from orange-spotted grouper (Epinephelus coioides) involved in viral infection.

Bcl-2-associated athanogene 5 (BAG5) is a kind of molecular chaperone that can bind to the Bcl-2 and modulate cell survival. However, little is known about the functions of fish BAG5. In this study, we characterized a BAG5 homolog from orange-spotted grouper (Epinephelus coioides) gene (Ec-BAG5) and investigated its roles during viral infection. The Ec-BAG5 protein encoded 468 amino acids with four BAG domains, which shared high identities with reported BAG5. The highest transcriptional level of Ec-BAG5 was found in the peripheral blood lymphocyte (PBL). And the Ec-BAG5 expression were significantly up-regulated after red-spotted grouper nervous necrosis virus (RGNNV) or Lipopolysaccharide (LPS) stimulation in vitro. Furthermore, Ec-BAG5 overexpression could inhibited viral replication and the expression of viral genes (coat protein (CP) and RNA-dependent RNA polymerase (RdRp)). Also, overexpression of Ec-BAG5 significantly increased the expression of interferon pathway-related factors including interferon regulatory factor 3 (IRF3), interferon-stimulated gene 15 (ISG15), interferon-induced protein 35 (IFP35), myxovirus resistance gene 1 (Mx1) and inflammatory-related factors including tumor necrosis factor receptor-associated factor 6 (TRAF6), tumor necrosis factor-α (TNF-α), interleukin-1 beta (IL-1ß), as well as the activities of NF-κB, ISRE and IFN-1. These data indicate that Ec-BAG5 can affect viral infection through regulating the expression of IFN- and inflammation-related factors, which provide useful information to better understand the immune response against viral infection.

Identification of Beclin-1 from orange-spotted grouper (Epinephelus coioides) involved in viral infection.

Beclin-1 is an essential autophagic regulator that plays diverse roles in physiology and disease. However, reports about the function of fish Beclin-1 during pathogen infection are still very limited. In this study, a Beclin-1 homolog (EcBeclin-1) from orange-spotted grouper (Epinephelus coioides) was identified and its roles in viral infection were investigated. EcBeclin-1 encoded 447amino acids protein with a BH3 domain, a CCD domain and an ECD domain, which shared high identities (97%-82%) with reported Beclin-1 proteins from mammal to fish. Quantitative real-time PCR (qRT-PCR) analysis revealed that EcBeclin-1 was predominantly expressed in brain and muscle of healthy grouper. Using fluorescence microscopy, we found that EcBeclin-1 was co-localized with endoplasmic reticulum (ER) in grouper spleen cells (EAGS). After red-spotted grouper nervous necrosis virus (RGNNV) infection in vitro, EcBeclin-1 transcript was significantly up-regulated, implying that EcBeclin-1 might be involved in viral infection. Furthermore, the in vitro studies of EcBeclin-1 overexpression promoted RGNNV induced autophagy, as well as the expression of coat protein (CP) and RNA-dependent RNA polymerase (RdRp). The overexpression of EcBeclin-1 suppressed the expressions of interferon pathway-related factors, inflammatory-related factors and activities of NF-κB and ISRE. Additionally, EcBeclin-1 could interact with EcBcl-xL in vitro. These data suggest that EcBeclin-1 affect viral replication through modulating IFN and inflammatory responses, as well as virus-induced cell death, which will help us to further explore the immune response of fish during viral infection.

[A preliminary exploration of fluid resuscitation during the shock stage of mass burn patients].

OBJECTIVE: To explore the effects of fluid resuscitation during the shock stage of mass burn patients. METHODS: Eleven major burn patients out of 25 were rescued at spot and were resuscitated. The treatment results were analyzed and concluded. RESULTS: The eleven patients were all cured by smoothly living through the shock stage. CONCLUSION: The approaching of medical personnel to the site might be the guarantee of correct and timely fluid resuscitation during shock stage, which could be effective on the prevention of burn shock development.