Epithelioma Papulosum Cyprinid Research Articles

Influence of heat shock protein 90 on the replication of spring viremia of carp virus

Spring viremia of the carp virus (SVCV) infection leads to a lethal acute hemorrhagic viral illness that causes substantial damage to the global aquaculture industry. Heat shock protein 90 (HSP90) is a highly conserved ATP-dependent molecular chaperone that plays important roles with intracellular proteins. Currently, little is known about the effect of HSP90 on SVCV replication. Therefore, in this study, we examined the transcription and expression levels of HSP90 in various organs of Cyprinus carpio var. Jian fish, including the brain, spleen, kidneys, liver, intestine, gills, heart, eyes, and muscles. We found that HSP90 was expressed at the highest levels in the liver, intestine, and gills and at the lowest levels in the spleen, eyes, and muscles. The SVCV-G and HSP90 levels in kidney, spleen, liver, and gill tissues peaked on day 3 following SVCV infection. We also constructed a recombinant HSP90 plasmid and demonstrated HSP90 overexpression in Epithelioma papulosum cyprinid (EPC) transfectants, using Pcs2 + 8CmCherry vector as a negative control. Laser confocal microscopy demonstrated that HSP90 was present in the cytoplasm of EPC cells. HSP90 expression gradually increased in EPC cells as the SVCV infection progressed. To gain insight into the influence of HSP90 on SVCV infection and its interconnectedness, we assessed apoptosis and immune gene expression by overexpressing and silencing HSP90. EPC cells infected with SVCV and overexpressing HSP90 showed considerably higher apoptosis, mortality, and IRF3 and IRF7 expression that EPC cells that were subjected to SVCV infection alone. HSP90 suppression increased the ability of cells to survive. Our initial investigation into the involvement of HSP90 in SVCV infection offers valuable insights. In addition, our findings offer novel approaches and techniques for constructing efficient systems to prevent and treat viral infections.

Morphogenesis of largemouth bass ranavirus (LMBRaV) in the epithelioma papulosum cyprinid cell line

Largemouth bass ranavirus (LMBRaV) belongs to the Ranavirus genus of the Iridoviridae family. It is a highly pathogenic virus that causes mass mortality in largemouth bass. In recent years, outbreaks of LMBRaV have been found in various provinces throughout China. Previous research mainly focused on virus isolation, identification, and detection, while the morphological change of the virus was still unknown. In this study, the ultrastructural morphogenesis of LMBRaV in epithelioma papulosum cyprinid (EPC) cells was observed and studied by using transmission electron microscopy. EPC cells were infected with LMBRaV (MOI=0.1) and then examined at 2 h, 4 h, 6 h, 12 h, 24 h, 48 h, 72 h and 96 h post infection. LMBRaV entered cells through endocytosis or direct penetration of cell membrane. After entering, the virus was observed in vesicles or lysosomes. After capsid uncoating, the virus genomes passed through the nuclear membrane and entered the cell nucleus. Virus genomes completed replication in the nucleus then transferred into the cytoplasm. In the cytoplasm, the progeny virus was assembled in the viromatrix and then aggregated in pseudocrystalline array. Finally, mature virus particles released through budding release from the cell membrane. Mature virus particles had a hexagonal shape and a diameter of approximately 150 nm. This study revealed the process of morphogenesis of LMBRaV in EPC cell line, providing essential information for further research on pathogenic mechanisms and immunological prevention of LMBRaV.

Isolation, Identification, and Pathogenicity Characterization of a Largemouth Bass Ranavirus from Fujian, China

This study aimed to isolate and characterize the pathogenic agent of a suspected viral disease of largemouth bass (Micropeterus salmoides), which broke out in an aquatic farm in Fujian, China. The diseased bass had ulcer skin and muscles, bleeding fin, lesion liver, and enlarged spleen. Histopathological analysis showed multifocal necrosis on the gill, hepatopancreas spleen, and kidney. In the epithelioma papulosum cyprinid (EPC) cell line, stable cytopathic effects (CPE) were observed. Electron microscopy analysis showed icosahedral virus particles in the cytoplasm with a 150-160 nm diameter. PCR amplification revealed that the isolated virus belongs to largemouth bass ranavirus (LMBV) and was designated as LMBV FJ_22109. Further phylogenetic analysis determined that LMBV FJ_22109 is a variant of European and American strains. The intraperitoneal injection of LMBV FJ_22109 resulted in a regress infection, demonstrating its ability to induce pathogenicity in largemouth bass. The cumulative mortality rate was 100% at a culture temperature of 29°C, and 47% at a culture temperature of 17°C. Thus, it confirmed that LMBV FJ_22109 is indeed the causative agent of the suspected viral disease, and its virulence could be influenced by temperature, which will be beneficial for preventing and managing the disease.

Potential to grow carp oedema virus (genogroup I) in monolayers of carp-derived primary cells with further implication in cell analysis.

Carp oedema virus (CEV) has distinct molecularly identified genogroups of viral mutations, denoted as I, IIa, and IIb. Failure to propagate CEV invitro limits studies towards understanding its interactions with host cells. Here, virus isolates belonging to genogroup I collected during natural outbreaks in the Czech Republic were employed for routine CEV cultivation in monolayers of carp-derived primary cells, common carp brain (CCB) cells, and epithelioma papulosum cyprinid (EPC) cells. Induction of cytopathic effects (CPEs) was observed and recorded in affected cells. Cell survival rate was evaluated under serial dilutions of the CEV inoculum. Virus cell entry was quantified and visualized by qPCR and transmission electron microscopy, respectively. Study findings indicate primary gills epithelia likely present the most suitable matrix for CEV growth invitro. Cells of the head kidney and spleen facilitate virus entry with microscopically confirmed CPEs and the presence of cytoplasmic pleomorphic virus particles. Cells of the trunk kidney and gonads are unlikely to permit virus cell entry and CPEs development. Although CEV cultivation in cell lines was inconclusive, EPC cells were CEV permissible. Monolayers of carp-derived primary cells show promise for CEV cultivation that could enable elaborate study of mechanisms underlying cellular binding and responses.

Rhein: A potent immunomodulator empowering largemouth bass against MSRV infection

Micropterus salmoides rhabdovirus (MSRV) is a significant viral pathogen in largemouth bass aquaculture, causing substantial annual economic losses. However, effective prevention methods remain elusive for various reasons. Medicinal plant extracts have emerged as valuable tools in preventing and managing aquatic animal diseases. Thus, the search for immunomodulators with straightforward, safe structures in plant extracts is imperative to ensure the continued health and growth of the largemouth bass industry. In our research, we employed epithelioma papulosum cyprinid (EPC) cells and largemouth bass as models to assess the anti-MSRV properties and immunomodulatory effects of ten plant-derived bioactive compounds. Among them, rhein demonstrated noteworthy potential, exhibiting a 75 % reduction in viral replication in vitro at a concentration of 50 mg/L. Furthermore, rhein pre-treatment significantly inhibited MSRV genome replication in EPC cells, with the highest inhibition rate reaching 64.8 % after 24 h, underscoring rhein's preventive impact against MSRV. Likewise, rhein displayed remarkable therapeutic effects on EPC cells during the early stages of MSRV infection, achieving a maximum inhibition rate of 85.6 % in viral replication. Subsequent investigations unveiled that rhein, with its consistent activity, effectively mitigated cytopathic effects (CPE) and nuclear damage induced by MSRV infection. Moreover, it restrained mitochondrial membrane depolarization and reduced the apoptosis rate by 38.8 %. In vivo experiments reinforced these findings, demonstrating that intraperitoneal injection of rhein enhanced the expression levels of immune related genes in multiple organs, hindered virus replication, and curtailed the mortality rate of MSRV-infected largemouth bass by 29 %. Collectively, our study endorses the utility of rhein as an immunomodulator to combat MSRV infections in largemouth bass. This not only underscores the potential of rhein as a broad-spectrum antiviral and means to bolster the immune response but also highlights the role of apoptosis as an immunological marker, making it an invaluable addition to the armamentarium against aquatic viral pathogens.

Transcriptome analysis reveals the relationship between Spring viremia of carp N protein-induced endoplasmic reticulum stress in Epithelioma papulosum cyprinid (EPC) cells and apoptosis and autophagy

Spring viremia of carp virus(SVCV) is one of the important pathogens of viral infectious diseases in fish, which brings severe economic losses to the aquaculture industry and is familiar and challenging to control viral disease in aquaculture. Currently, studies have shown that SVCV can induce endoplasmic reticulum stress, whereas the relationship between whether the most abundant protein of SVCV-N, can induce endoplasmic reticulum stress, autophagy and apoptosis has not been reported. In this study, the SVCV-N protein was overexpressed in EPC cells. According to transcriptome analysis of gene changes in EPC cells, there were 2290 differentially expressed genes, 1137 up-regulated and 1153 down-regulated genes compared to controls transfected with the empty vector pCS 2 + 8CmCherry. After gene ontology (GO) analysis, they were classified into three categories: biological processes, molecular functions and cellular components. KEGG enrichment analysis showed that 722 differentially expressed genes were enriched in 190 KEGG pathways, most of them were enriched in endoplasmic reticulum processing. Western blotting showed that GRP78, Beclin1 and Lc3-II/Lc3- I protein expression was increased, and p62 protein expression was decreased. Flow cytometry showed an increased rate of apoptosis. Activation inhibition experiments showed that activation of endoplasmic reticulum stress expression by SVCV-N protein promotes autophagy and apoptosis, and vice versa. The qRT-PCR results are consistent with the RNA-Seq, indicating that the transcriptome sequencing data are reliable. This study provides preliminary insight into the pathogenic mechanism of SVCV-N infection and provides data to reveal the mechanism.

Spring Viremia of Carp Virus Infection Induces Carp IL-10 Expression, Both In Vitro and In Vivo.

Interleukin-10 (IL-10) is a pleiotropic cytokine with both immune enhancement and immunosuppression activities, but the main role is immunosuppression and anti-inflammatory ability. In order to use the immunosuppressive function of IL-10, many viruses, such as SARS-CoV-2, hepatitis B virus and EB virus, can evade the host's immune surveillance and clearance by increasing the expression of host IL-10. However, it has not been reported whether the aquatic animal infection virus can upregulate the expression of host IL-10 and the mechanisms are still unknown. Spring viremia of carp (SVC) is a fatal viral disease for many fish species and is caused by spring viremia of carp virus (SVCV). This disease has caused significant economic losses in the aquaculture industry worldwide. In this study, the expression of carp IL-10 with or without infection of SVCV in epithelioma papulosum cyprinid (EPC) cells, carp head kidney (cHK) primary cells and common carp tissues were analyzed using RT-PCR and ELISA. The results show that SVCV infection induced carp IL-10 mRNA and protein expression, both in vitro and in vivo. However, the upregulation of carp IL-10 by SVCV was hindered by specific inhibitors of the JAK inhibitor (CP-690550), STAT3 inhibitor (STA-21), NF-κB inhibitor (BAY11-7082) and p38 MAPK (mitogen-activated protein kinase) inhibitor (SB202190), but not JNK inhibitor (SP600125). Furthermore, the results demonstrated that JAK1, JAK2, JAK3, TYK2 and STAT5 played important roles in carp IL-10 production induced by SVCV infection. Taken together, SVCV infection significantly induced carp IL-10 expression and the upregulation trigged in JAK-STAT, NF-κB and p38MAPK pathways. To our knowledge, this is the first time that a fish infection virus upregulated the host IL-10 expression through the JAK-STAT, NF-κB and p38MAPK pathways. Altogether, fish viruses may have a similar mechanism as human or other mammalian viruses to escape host immune surveillance and clearance.

Micropterus salmoides rhabdovirus enters cells via clathrin-mediated endocytosis pathway in a pH-, dynamin-, microtubule-, rab5-, and rab7-dependent manner.

Although Micropterus salmoides rhabdovirus (MSRV) causes serious fish epidemics worldwide, the detailed mechanism of MSRV entry into host cells remains unknown. Here, we comprehensively investigated the mechanism of MSRV entry into epithelioma papulosum cyprinid (EPC) cells. This study demonstrated that MSRV enters EPC cells via a low pH, dynamin-dependent, microtubule-dependent, and clathrin-mediated endocytosis. Subsequently, MSRV transports from early endosomes to late endosomes and further into lysosomes in a microtubule-dependent manner. The characterization of MSRV entry will further advance the understanding of rhabdovirus cellular entry pathways and provide novel targets for antiviral drug against MSRV infection.

The role of CcPTGS2a in immune response against Aeromonas hydrophila infection in common carp (Cyprinus carpio)

Prostaglandin-endoperoxide synthase 2 (PTGS2), a crucial enzyme in prostaglandin synthesis, catalyzes the conversion of arachidonic acid to prostaglandins and plays a significant role in the inflammatory response. This investigation aimed to determine the regulatory role of PTGS2a in the innate immune response to bacterial infection in fish. To achieve this objective, the CcPTGS2a gene was identified and characterized in common carp (Cyprinus carpio), and its function in immune defense was investigated. According to the sequence and structural analysis results, CcPTGS2a had an open reading frame of 1806 bp that encoded 602 amino acids. It was estimated that the protein's theoretical molecular weight was 69.0 kDa, and its isoelectric point was 8.10. The structure of CcPTGS2a was observed to be conserved, with an epidermal growth factor domain and a peroxidase domain present. Moreover, the amino acid sequence of CcPTGS2a exhibited significant homology with the amino acid sequences of several fish species. CcPTGS2a mRNA was detected in the healthy tissues of common carp, with higher expression in the head kidney, spleen, gills, and liver. Following the challenges with Aeromonas hydrophila and lipopolysaccharide, CcPTGS2a mRNA showed unique geographic and temporal expression patterns, with significant increases detected in the head kidney, gills, spleen, and liver. Additionally, the recombinant CcPTGS2a protein exhibited detectable bacterial binding to various bacteria. As determined by subcellular localization analysis, CcPTGS2a was predominantly localized in the nucleus and cytoplasm. Furthermore, it was discovered that the overexpression of CcPTGS2a stimulated the up-regulation of ferroptosis-related genes and inflammatory cytokine mRNA expression in fish and EPC (Epithelioma papulosum cyprinid) cells while concurrently reducing the bacterial load of A. hydrophila. In contrast, the interference of CcPTGS2a decreased the mRNA expression of ferroptosis-related genes and inflammatory cytokines in fish and EPC cells and increased the bacterial load of A. hydrophila. Notably, A. hydrophila stimulation resulted in the up-regulation of CcPTGS2a protein expression in EPC cells. These results suggested that CcPTGS2a was involved in the immune response to bacterial infections in common carp.

Activation Mechanism of CcGSDMEb-1/2 and Regulation for Bacterial Clearance in Common Carp (Cyprinus carpio).

Gasdermin E (GSDME), to date, is considered the only direct executor of the pyroptosis process in teleost and plays an important role in innate immunity. In common carp (Cyprinus carpio), there contains two pairs of GSDME (GSDMEa/a-like and GSDMEb-1/2), and the pyroptotic function and regulation mechanism of GSDME still remain unclear. In this study, we identified two GSDMEb genes of common carp (CcGSDMEb-1/2), which contain a conserved N-terminal pore-forming domain, C-terminal autoinhibitory domain, and a flexible and pliable hinge region. We investigated the function and mechanism of CcGSDMEb-1/2 in association with inflammatory and apoptotic caspases in Epithelioma papulosum cyprinid cells and discovered that only CcCaspase-1b could cleave CcGSDMEb-1/2 through recognizing the sites 244FEVD247 and 244FEAD247 in the linker region, respectively. CcGSDMEb-1/2 exerted toxicity to human embryonic kidney 293T cells and bactericidal activity through its N-terminal domain. Interestingly, after i.p. infection by Aeromonas hydrophila, we found that CcGSDMEb-1/2 were upregulated in immune organs (head kidney and spleen) at the early stage of infection, but downregulated in mucosal immune tissues (gill and skin). After CcGSDMEb-1/2 were knocked down and overexpressed invivo and invitro, respectively, we found that CcGSDMEb-1/2 could govern the secretion of CcIL-1β and regulate the bacterial clearance after A. hydrophila challenge. Taken together, in this study, it was demonstrated that the cleavage mode of CcGSDMEb-1/2 in common carp was obviously different from that in other species and played an important role in CcIL-1β secretion and bacterial clearance.

Inhibition of the largemouth bass virus replication by piperine demonstrates potential application in aquaculture

Largemouth bass virus (LMBV) is a systemic viral pathogen that can cause high mortality rates in cultivated largemouth bass. However, no treatment is currently approved. Therapeutic strategies against LMBV infection are urgently needed. In this study, we investigated the antiviral activity of piperine against LMBV in vitro and in vivo. In vitro antiviral activity assay showed that 210.28 μM piperine significantly decreased LMBV major capsid protein (MCP) gene expression in epithelioma papulosum cyprinid (EPC) cells by a maximum inhibitory rate of >95%. Piperine treatment inhibited LMBV replication in a dose-dependent manner, with the half-maximal activity (IC50 ) of 34.61 μM. Moreover, piperine significantly decreased the viral titers and cytopathic effects (CPE), contributing to the protection of infected cells. With regard to the steps of piperine affecting the life cycle of viruses, piperine had a direct inactivating effect on LMBV. During the virus adsorption phase, piperine prevented the adsorption of LMBV to EPC cells. Furthermore, piperine played an antiviral role mainly in the later stages of viral infection (4-8h). To further evaluate the antiviral activity of piperine against LMBV in vivo, largemouth bass as a model organism was carried out in relevant experiments. Intraperitoneal injection of piperine (25 mg/kg) effectively improved the survival rate of LMBV-infected largemouth bass by 20%. In addition, RT-qPCR results of viral replication in liver, spleen, kidney, gill and swim bladder tissues showed that piperine significantly inhibited LMBV replication in vivo, thus protecting largemouth bass from LMBV-induced death. Together, our results suggested that piperine is a therapeutic and preventative agent against LMBV infection.

Toxic effects of glyphosate on the intestine, liver, brain of carp and on epithelioma papulosum cyprinid cells: Evidence from in vivo and in vitro research.

Glyphosate (GLY) is the most widely used organophosphorus herbicide in agriculture. The present study aimed to analyze the comprehensive toxicological effects of GLY on juvenile common carp and an epithelioma papulosum cyprinid (EPC) cell line. In the in vivo experiments, exposure to GLY (5 and 15mg/L) for 30 days induced liver inflammation and oxidative damage in common carp and changed the physical barrier of the intestine. Histopathological analysis of the intestine, liver, brain, and changes in oxidative stress biomarkers provided evidence of damage and immune system responses to GLY. Moreover, an inhibitory effect of 15mg/L GLY on acetylcholinesterase (AChE) activity was found in the brain, which may be an important reason for the significant decrease in both swimming distance and average acceleration of common carp. Cell experiments showed that 0.65 and 3.25mg/L GLY inhibited the viability of EPCs. Furthermore, oxidative DNA damage, mitochondrial dysfunction, and reactive oxygen species (ROS) production were observed in EPC cells following GLY exposure. Taken together, this study not only highlights the negative effects of GLY on common carp but also enriches the knowledge of the cytotoxicity mechanism to further clarify the comprehensive toxicity of GLY in common carp.

Evaluation on the antiviral activity of artemisinin against rhabdovirus infection in common carp

The spring viremia of carp virus (SVCV) is a serious deadly virus that results in high mortality and significant economic losses in the aquaculture industry of carp species. The present study was conducted to evaluate the antiviral effect of 8 natural compounds against SVCV in epithelioma papulosum cyprinid (EPC) cells and common carp (Cyprinus carpio). Herein, artemisinin was identified to inhibit SVCV replication obviously, with the highest inhibition ratio on SVCV nucleoprotein gene expression at 100 μM in cells. Artemisinin not only maintained normal cell morphology and reduced intracellular nuclear damage in SVCV-infected cells, but also disrupted SVCV infection by affecting viral attachment and entry. In vivo experiments revealed that artemisinin post-incubation by feeding significantly increased common carp survival by 33.3%, and significantly decreased the viral loads. Meanwhile, artemisinin induced up-regulation of antiviral-related genes, such as type I IFN (IFN1), IFN-stimulated gene product 15 (ISG15), Mx1, and viperin, implying an immunoregulatory capacity. Taking together, artemisinin may promise in the prevention and treatment of SVCV infection in aquaculture.

In vitro and in vivo inhibition of a novel arctigenin derivative on aquatic rhabdovirus

Spring viraemia of carp virus (SVCV) poses a serious threat to aquaculture industry due to the lack of approved antiviral treatments. Therefore, a novel arctigenin derivative, 4-(2-methylimidazole) octanoxy-arctigenin (MON), was synthesized to assess the antiviral activity against SVCV in vitro and in vivo. The results indicated MON decreased the SVCV glycoprotein (G) gene expression in vitro by a maximum inhibitory rate of > 99% at 3.5 μM. Furthermore, MON showed the protective effect on epithelioma papulosum cyprinid (EPC) cells and considerably decreased the cytopathic effect (CPE). More importantly, MON inhibited SVCV G gene expression levels in vitro at the half-maximal activity (IC50) of 0.18 μM at 48 h. For in vivo studies, MON demonstrated anti-SVCV activity by enhancing the survival rate of zebrafish (Danio rerio) after infection via pelvic fin base injection. These results tended to be consistent with MON decreasing the SVCV titer of infected zebrafish. During this time, viral loads of the spleen and kidney have declined in SVSV-infected zebrafish. Based on the histopathological assay, MON exhibited the high protective effect in the spleen and kidney of SVCV-infected fish. Combined, MON is on track to become a novel agent to address SVCV infection in aquaculture.

Autophagy induced by largemouth bass virus inhibits virus replication and apoptosis in epithelioma papulosum cyprini cells

Autophagy and apoptosis play important roles in the occurrence and development of diseases. Largemouth bass virus (LMBV) is a primary agent that causes infectious skin ulcerative syndrome in largemouth bass and threatens the aquaculture of the species. We investigated the relationship between LMBV and autophagy, as well as the effect of autophagy on apoptosis induced by LMBV. Results showed that LMBV could induce autophagy in epithelioma papulosum cyprinid (EPC) cells. There was also an increase in LC3-II protein and decrease in p62 protein, along with autophagosome-like membranous vesicles and punctate autophagosomes fluorescent spots being observed in EPC cells. Enhancing autophagy inhibited the replication of LMBV and apoptosis in EPC cells while inhibiting autophagy produced the opposite effect. These results offer new insights into the pathogenesis of LMBV and anti-LMBV strategies.

Antiviral effects of natural small molecules on aquatic rhabdovirus by interfering with early viral replication.

Spring viremia of carp virus (SVCV) is globally widespread and poses a serious threat to aquatic ecology and aquaculture due to its broad host range. To develop effective agents to control SVCV infection, we selected 16 naturally active small molecules to assess their anti-SVCV activity. Notably, dihydroartemisinin (DHA) (100 µmol/L) and (S, S)-(+)-tetrandrine (TET) (16 µmol/L) exhibited high antiviral effects in epithelioma papulosum cyprinid (EPC) cells, with inhibitory rates of 70.11% and 73.54%, respectively. The possible antiviral mechanisms were determined as follows: 1. Pre-incubation with DHA and TET decreased viral particle infectivity in fish cells, suggesting that horizontal transmission of SVCV in the aquatic environment was disrupted; 2. Although neither had an effect on viral adhesion, TET (but not DHA) interfered with SVCV entry into host cells (>80%), suggesting that TET may have an antiviral function in early viral replication. For in vivo study, both agents enhanced the survival rate of SVCV-infected zebrafish by 53.3%, significantly decreased viral load, and modulated the expression of antiviral-related genes, indicating that DHA and TET may stimulate the host innate immune response to prevent viral infection. Overall, our findings indicated that DHA and TET had positive effects on suppressing SVCV infection by affecting early-stage viral replication, thus holding great potential as immunostimulants to reduce the risk of aquatic rhabdovirus disease outbreaks.

Evaluation of the effects of Astragalus polysaccharides as immunostimulants on the immune response of crucian carp and against SVCV in vitro and in vivo

This experiment was conducted to evaluate the immunomodulatory effect and antiviral activity of Astragalus polysaccharides (APS) in crucian carp and epithelioma papulosum cyprinid (EPC) cells. Two diets containing 0 and 2 g/kg, APS were fed crucian carp for 56 days. The results showed that supplementation with APS significantly upregulated the immune-related indices including the levels of IgM, the activities of LZM, AKP and ACP, and the contents of C3 and C4. At the same time, compared with the CK group, adding APS to the feed significantly upregulated the expression of IL-8, IL-10, IL-1β, IFN-α, IFN-γ, MyD88, TGF-β and TNF-α in the spleen, kidney, liver and intestine of crucian carp. In addition, when the crucian carp were injected with SVCV, the survival rates of fish in the APS group and the control group were 48.87% and 13.76%, respectively. These results indicated that dietary APS could improve the resistance of crucian carp against SVCV infection. APS also significantly decreased viral titer and inhibited apoptosis induced by SVCV in EPC cells. These results indicated that APS could stimulate the immune response of crucian carp and improve the abilities of crucian carp and EPC cells to resist SVCV infection.

Synthesis and biological evaluation of novel coumarin derivatives in rhabdoviral clearance.

Diseases caused by rhabdoviruses have had a huge impact on the productive lives of the entire human population. The main problem is the lack of drugs for the treatment of this family of viruses. Infectious hematopoietic necrosis virus (IHNV), the causative agent of IHN, is a typical rhabdovirus which has caused huge losses to the salmonid industry. Therefore, in this study, IHNV was studied as a model to evaluate the antiviral activity of 35 novel coumarin derivatives. Coumarin A9 was specifically selected for further validation studies upon comparing the half maximum inhibitory concentration (IC50) of four screened candidate derivatives in epithelioma papulosum cyprinid (EPC) cells, as it exhibited an IC50 value of 2.96μM against IHNV. The data revealed that A9 treatment significantly suppressed the virus-induced cytopathic effect (CPE) in EPC cells. In addition, A9 showed IC50 values of 1.68 and 2.12μM for two other rhabdoviruses, spring viremia of carp virus and micropterus salmoides rhabdovirus, respectively. Furthermore, our results suggest that A9 exerts antiviral activity, but not by destroying the virus particles and interfering with the adsorption of IHNV. Moreover, we found that A9 had an inhibitory effect on IHNV-induced apoptosis in EPC cells, as reflected by the protection against cell swelling, formation of apoptotic bodies, and loss of cell morphology and nuclear division. There was a 19.05% reduction in the number of apoptotic cells in the A9 treatment group compared with that in the IHNV group. In addition, enzyme activity assays proved that A9 suppressed the expression of caspase 3, 8 and 9. These results suggested that A9 inhibit viral replication, to some extent, by blocking IHNV-induced apoptosis. In an invivo study, A9 exhibited an anti-rhabdovirus effect in virus-infected fish by substantially enhancing the survival rate. Consistent with the above results, A9 repressed IHNV gene expression in virus-sensitive tissues (brain, kidney and spleen) in the early stages of virus infection. Importantly, the data showed that horizontal transmission of IHNV was reduced by A9 in a static cohabitation challenge model, especially in fish that underwent bath treatment, suggesting that A9 might be a suitable therapeutic agent for IHNV in aquaculture. Therefore, coumarin derivatives can be developed as antiviral agents against rhabdoviruses.

ATG12 is involved in the antiviral immune response in large yellow croaker (Larimichthys crocea)

ATG12, a core autophagy protein, forms a conjugate with ATG5 to promote the formation of autophagosome membrane, and plays an important role in antiviral immunity. However, little is known about the function of ATG12 in fish. Here, we cloned the open reading frame (ORF) of large yellow croaker (Larimichthys crocea) ATG12 (LcATG12), which is 354 nucleotides long and encodes a protein of 117 amino acids. The deduced LcATG12 possesses a conserved APG12 domain (residues 31 to 117), and shares 91.45% identities with ATG12 in orange-spotted grouper (Epinephelus coioides). LcATG12 was constitutively expressed in all examined tissues, with the highest level in intestine. Its transcript was also detected in primary head kidney granulocytes (PKG), primary head kidney macrophages (PKM), primary head kidney lymphocytes (PKL), and large yellow croaker head kidney (LYCK) cell line, and was significantly up-regulated by poly(I:C). LcATG12 was regularly distributed in both cytoplasm and nucleus of LYCK and epithelioma papulosum cyprinid (EPC) cells. Overexpression of LcATG12 in EPC cells significantly inhibited the replication of spring viremia of carp virus (SVCV). Further studies reveled that LcATG12 could induce the occurrence of autophagy in LYCK cells. Furthermore, overexpression of LcATG12 in LYCK cells increased the expression levels of large yellow croaker type I interferons (IFNs, IFNc, IFNd, and IFNh), IFN regulatory factors (IRF3 and IRF7), and IFN-stimulated genes (PKR, Mx, and Viperin). All these data indicated that LcATG12 plays a role in the antiviral immunity possibly by inducing both autophagy and type I IFN response in large yellow croaker.

Autophagy Delays Apoptotic Cell Death Induced by Siniperca chuatsi Rhabdovirus in Epithelioma Papulosum Cyprinid Cells.

Autophagy and apoptosis are two key cell fate determination pathways, which play vital roles in the interaction between viruses and host cells. Previous research had confirmed that one strain of fish rhabdoviruses, Siniperca chuatsi rhabdovirus (SCRV), could induce apoptosis and autophagy after infection. In the current study, we continued to analyze the interaction of autophagy and apoptosis in SCRV-infected EPC cell lines after treatment with different autophagy or apoptosis inhibitors. We found that SCRV infection could activate the mitochondrial apoptotic pathway by the detection of the activities of the caspase-3 and caspase-9 and by flow cytometry analysis in JC-1-stained cells, respectively. Furthermore, no significant autophagy-related factors were disturbed in SCRV-infected cell after apoptosis inhibitor Z-VAD-FMK treatment, while autophagy inducer rapamycin could obviously delay the occurrence of CPE and cell death. Meanwhile, rapamycin was able to reduce the proportion of apoptotic cells. Besides that, rapamycin could disturb the expression of p62 and LC3B-II, and the transcription level of SCRV nucleoprotein mRNA. The progeny virus titers did not show a big difference between the rapamycin treatment or without it. Collectively, our data preliminarily confirmed that SCRV-activated autophagy could delay apoptosis in EPC cells and may not affect virus production. Further study may need to focus on the crosstalk regulation and its roles on the SCRV infection.