Singapore Grouper Iridovirus Infection Research Articles

Evaluation of protective immune response of immersion inactivated vaccine against Singapore grouper iridovirus

Singapore grouper iridovirus (SGIV) always causes high transmission efficiency and mortality in the larval and juvenile stages of grouper in aquaculture industry. Although inactivated virus and recombinant DNA vaccines administered via intraperitoneal injection have shown efficacy in protection against SGIV, their potential applications in field testing were limited due to the vaccine delivery methods. Here, we developed an immersion vaccine containing inactivated virus and Montanide IMS 1312 adjuvant (IMS 1312) and evaluated its protective efficacy against SGIV infection. Compared to the PBS group, fish vaccinated with immersion inactivated vaccine with or without IMS 1312 were significantly protected against SGIV, with a relative percent survival (RPS) of 57.69 % and 38.47 %, respectively. Furthermore, the transcripts of viral core genes were reduced, and the histopathological severity caused by SGIV were relatively mild in multiple tissues of the IMS + V group. The immersion vaccine activated the AKP and ACP activities and increased the mRNA levels of IFN and inflammation-associated genes. The transcriptome analysis showed that a total of 731 and 492 genes were significantly regulated in the spleen and kidney from the IMS + V group compared to the PBS group, respectively. Among them, 129 DEGs were co-regulated, and enriched in the KEGG pathways related to immune and cell proliferation, including MAPK signaling, JAK-STAT signaling and PI3K-Akt signaling pathways. Similarly, the DEGs specially regulated in the kidney and spleen upon vaccine immunization were significantly enriched in the KEGG pathways related to interferon and inflammation response. Together, our results elucidated that the immersion vaccine of inactivated SGIV with IMS 1312 induced a protective immune response of grouper against SGIV.

Epinephelus coioides Sec3 promotes Singapore grouper iridovirus infection by negatively regulates immune response

Exocyst, a protein complex, plays a crucial role in various cellular functions, including cell polarization, migration, invasion, cytokinesis, and autophagy. Sec3, known as Exoc1, is a key subunit of the Exocyst complex and can be involved in cell survival and apoptosis. In this study, two subtypes of Sec3 were isolated from Epinephelus coioides, an important marine fish in China. The role of E. coioides Sec3 was explored during Singapore grouper iridovirus (SGIV) infection, an important pathogen of marine fish which could induce 90 % mortality. E. coioides Sec3 sequences showed a high similarity with that from other species, indicating the presence of a conserved Sec3 superfamily domain. E. coioides Sec3 mRNA could be detected in all examined tissues, albeit at varying expression levels. SGIV infection could upregulate E. coioides Sec3 mRNA. Upregulated Sec3 significantly promoted SGIV-induced CPE, and the expressions of viral key genes. E. coioides Sec3 could inhibit the activation of NF-κB and AP-1, as well as SGIV-induced cell apoptosis. The results illustrated that E. coioides Sec3 promotes SGIV infection by regulating the innate immune response.

In vitro antiviral activity of eugenol on Singapore grouper iridovirus

The high mortality rate of Singapore grouper iridovirus (SGIV) posing a serious threat to the grouper aquaculture industry and causing significant economic losses. Therefore, finding effective drugs against SGIV is of great significance. Eugenol (C10H12O2) is a phenolic aromatic compound, has been widely studied for its anti-inflammatory, antioxidant and antiviral capacity. In this study, we explored the effect of eugenol on SGIV infection and its possible mechanisms using grouper spleen cells (GS) as an in vitro model. We found that treatment of GS cells with 100 μM eugenol for 4 h exhibited the optimal inhibitory effect on SGIV. Eugenol was able to reduce the expression level of inflammatory factors by inhibiting the activation of MAPK pathway and also inhibited the activity of NF-κB and AP-1 promoter. On the other hand, eugenol attenuated cellular oxidative stress by reducing intracellular ROS and promoted the expression of interferon-related genes. Therefore, we conclude that eugenol inhibits SGIV infection by enhancing cellular immunity through its anti-inflammatory and antioxidant functions.

Identification and functional characterization of interleukin-22 (IL-22) in orange-spotted grouper (Epinephelus coioides)

In mammals, IL-22 is considered as a critical cytokine regulating of immunity and homeostasis at barrier surfaces. Although IL-22 have been functional characterization in different species of fish, the studies about distinct responses of IL-22 in different organs/tissues/cell types is rather limited. Here, we identified and cloned IL-22 gene (named as Ec-IL-22) from grouper (Epinephelus coioides). Ec-IL-22 gene was detected in all orangs/tissues examined, and was induced in intestine, gill, spleen, head kidney, and primary head kidney/intestine leukocytes following the stimulation of LPS and poly (I:C), as well as Vibrio harveyi and Singapore grouper iridovirus infection (SGIV). In addition, the stimulation of DSS could induce the expression of Ec-IL-22 in intestine and primary leukocytes from intestine. Importantly, the treatment of recombinant Ec-IL-22 induced the mRNA level of proinflammatory cytokines in primary intestine/head kidney leukocytes. The present results improve the understanding of expression patterns and functional characteristics of fish IL-22 in different organs/tissues/cell types.

Fish ELOVL7a is involved in virus replication via lipid metabolic reprogramming

The elongation of very long chain fatty acids (ELOVL) proteins are key rate-limiting enzymes that catalyze fatty acid synthesis to form long chain fatty acids. ELOVLs also play regulatory roles in the lipid metabolic reprogramming induced by mammalian viruses. However, little is known about the roles of fish ELOVLs during virus infection. Here, a homolog of ELOVL7 was cloned from Epinephelus coioides (EcELOVL7a), and its roles in red-spotted grouper nervous necrosis virus (RGNNV) and Singapore grouper iridovirus (SGIV) infection were investigated. The transcription level of EcELOVL7a was significantly increased upon RGNNV and SGIV infection or other pathogen-associated molecular patterns stimulation in grouper spleen (GS) cells. Subcellular localization analysis showed that EcELOVL7a encoded an endoplasmic reticulum (ER) related protein. Overexpression of EcELOVL7a promoted the viral production and virus release during SGIV and RGNNV infection. Furthermore, the lipidome profiling showed that EcELOVL7a overexpression reprogrammed cellular lipid components in vitro, evidenced by the increase of glycerophospholipids, sphingolipids and glycerides components. In addition, VLCFAs including FFA (20:2), FFA (20:4), FFA (22:4), FFA (22:5) and FFA (24:0), were enriched in EcELOVL7a overexpressed cells. Consistently, EcELOVL7a overexpression upregulated the transcription level of the key lipid metabolic enzymes, including fatty acid synthase (FASN), phospholipase A 2α (PLA 2α), and cyclooxygenases −2 (COX-2), LPIN1, and diacylglycerol acyltransferase 1α (DGAT1α). Together, our results firstly provided the evidence that fish ELOVL7a played an essential role in SGIV and RGNNV replication by reprogramming lipid metabolism.

Berberine inhibits SGIV replication by suppressing inflammatory response and oxidative stress

Singapore grouper iridovirus (SGIV) is one of the major infectious diseases responsible for high mortality and huge economic losses in the grouper aquaculture industry. Berberine (BBR), a naturally occurring plant alkaloid, is a phytochemical having a variety of biological properties, such as antiviral, antioxidant, and anti-inflammatory effects. In this work, we used an in vitro model based on Western blot, ROS fluorescence probe, and real-time quantitative PCR (qRT-PCR) to examine the antiviral qualities of BBR against SGIV. The outcomes demonstrated that varying BBR concentrations could significantly inhibit the replication of SGIV. In addition, BBR greatly inhibited the production of genes associated with pro-inflammatory cytokines in SGIV-infected or SGIV-uninfected GS cells based on qRT-PCR data. Subsequent investigations demonstrated that BBR suppressed the expression of the promoter activity of NF-κB and NF-κB-p65 protein. Additionally, BBR reduced the phosphorylation of ERK 1/2, JNK, and p38. Furthermore, BBR also inhibits SGIV-induced ROS production by upregulating the expression of antioxidant-related genes. In conclusion, BBR is a viable therapy option for SGIV infection due to its antiviral properties.

Singapore grouper iridovirus VP20 interacts with grouper TBK1 and IRF3 to attenuate the interferon immune response

Singapore grouper iridovirus (SGIV), belonging to genus Ranavirus, family Iridoviridae, is a highly pathogenic agent and causes heavy economic losses in the global grouper aquaculture. Recent studies demonstrated that SGIV infection attenuated antiviral immune and inflammatory response induced by poly (I:C) in vitro. However, little was known about the potential functions of the immune regulatory proteins encoded by SGIV. Here, we identified the detailed roles of VP20 and clarified the potential mechanism underlying its immune regulatory function during SGIV infection. Our results showed that VP20 was an IE gene, and partially co-localized with Golgi apparatus and lysosomes in grouper cells. Overexpression of VP20 enhanced SGIV replication, demonstrated by the increase in the transcription levels of viral core genes and the protein synthesis of MCP. Reporter gene assays showed that SGIV VP20 overexpression significantly reduced the IFN promoter activity induced by poly (I:C), grouper stimulator of interferon genes (EcSTING) and TANK-binding kinase 1 (EcTBK1). Consistently, the transcription levels of IFN related genes were significantly decreased in VP20 overexpressing cells compared to those in control cells. Co-IP assay and confocal microscopy observations indicated that VP20 co-localized and interacted with EcTBK1 and EcIRF3, but not EcSTING. In addition, VP20 was able to degrade EcIRF3 and attenuate the antiviral action of EcIRF3, while had no effect on EcTBK1. Together, SGIV VP20 was speculated to promote viral replication through attenuating the IFN response mediated by TBK1-IRF3 in vitro. Our findings provided new insights into the immune regulatory function of SGIV encoded unknown proteins.

Antiviral Effect and Mechanism of Edaravone against Grouper Iridovirus Infection.

Singapore grouper iridovirus (SGIV) is a virus with high fatality rate in the grouper culture industry. The outbreak of SGIV is often accompanied by a large number of grouper deaths, which has a great impact on the economy. Therefore, it is of great significance to find effective drugs against SGIV. It has been reported that edaravone is a broad-spectrum antiviral drug, most widely used clinically in recent years, but no report has been found exploring the effect of edaravone on SGIV infections. In this study, we evaluated the antiviral effect of edaravone against SGIV, and the anti-SGIV mechanism of edaravone was also explored. It was found that the safe concentration of edaravone on grouper spleen (GS) cells was 50 µg/mL, and it possessed antiviral activity against SGIV infection in a dose-dependent manner. Furthermore, edaravone could significantly disrupt SGIV particles and interference with SGIV binding to host cells, as well as SGIV replication in host cells. However, edaravone was not effective during the SGIV invasion into host cells. This study was the first time that it was determined that edaravone could exert antiviral effects in response to SGIV infection by directly interfering with the processes of SGIV infecting cells, aiming to provide a theoretical basis for the control of grouper virus disease.

Identification of circRNAs and circRNA-mRNA network of Epinephelus coioides during Singapore grouper iridovirus infection

Circular RNA (circRNA), one of the important non-coding RNA molecules with a closed-loop structure, plays a key regulatory role in cell processing. In this study, circRNAs of Epinephelus coioides, an important marine cultured fish in China, were isolated and characterized, and the network of circRNAs and mRNA was explored during Singapore grouper iridovirus (SGIV) infection, one of the most important double stranded DNA virus pathogens of marine fish. 10 g of raw data was obtained by high-throughput sequencing, and 2599 circRNAs were classified. During SGIV infection, 123 and 37 circRNAs occurred differential expression in spleen and spleen cells, indicating that circRNAs would be involved in the viral infection. GO annotation and KEGG demonstrated that circRNAs could target E. coioides genes to regulate cell activity and the activation of immune factors. The results provide some insights into the circRNAs mediated immune regulatory network during bony fish virus infection.

Lcalmnb2: Asian seabass gene involved in resisting SGIV

Diseases cause significant economic loss in aquaculture. Molecular breeding is an effective way to improve long-lasting disease resistance. DNA markers associated with disease resistance are essential in molecular breeding. The Singapore grouper iridovirus (SGIV) causes severe mortality in hatcheries of Asian seabass (Lates calcarifer). To identify DNA markers associated with SGIV resistance and understand more about functions of candidate genes in SGIV resistance, the lmnb2 gene from L. calcarifer was characterized. The gene contained 13 exons and 12 introns. Its open reading frame (ORF) of 1731 bp encoded a protein with 576 amino acids. A SNP, identified in the first intron of the gene, was significantly associated with SGIV resistance. Lcalmnb2 was ubiquitously expressed in organs of the healthy Asian seabass. In vitro studies revealed that Lcalmnb2 promoted cell proliferation and migration. Overexpression of the gene repressed the replication of SGIV in Asian seabass cells, while knockdown of the gene prompted SGIV replication. In virus-infected cells, SGIV utilized Lamin B2 for host DNA transport during viral replication, leading to the induction of nuclear abnormalities. This study sheds new light on the involvement of Lcalmnb2 in resisting SGIV infection. The identified SNP may serve as a useful DNA marker for selecting SGIV-resistant Asian seabass individuals in future breeding programs.

Taurochenodeoxycholic acid exerts anti-viral activities upon SGIV infection via anti-inflammatory response

Taurine chenodeoxycholic acid (TCDCA), a combination of taurine and chenodeoxycholic acid, plays anti-inflammatory roles in many diseases. However, little is known about its antiviral activity in aquaculture. Singapore grouper iridovirus (SGIV), a novel member of the genus Ranavirus, is an important viral pathogen in grouper aquaculture. Here, the effects of TCDCA on SGIV replication in vitro and in vivo were investigated. TCDCA treatment inhibited SGIV infection in grouper spleen (GS) cells, evidenced by the delayed CPE progression and decreased viral gene transcription, protein expression and viral progeny production. TCDCA treatment suppressed the mRNA level of pro-inflammatory factors and NF-κB promoter activities in SGIV-infected cells compared with mock-infected cells. Inhibition of NF-κB activity enhanced the inhibitory effects of TCDCA on SGIV replication, indicating that TCDCA inhibited SGIV replication in vitro via regulating host inflammatory response. Furthermore, TCDCA treatment protected grouper from SGIV infection, evidenced by the decreased mortality, the down-regulated transcription levels of viral genes and the improved pathological symptoms. In addition, the expression levels of SGIV induced pro-inflammation factors were decreased, while those of the antioxidant genes were increased after the supplement with TCDCA. Thus, our findings will not only contribute greatly to understanding the mechanism of SGIV pathogenesis, but also demonstrate that TCDCA might be a potential antiviral agent against SGIV infection.

The Role of Epinephelus coioides DUSP5 in Regulating Singapore Grouper Iridovirus Infection.

The dual-specificity phosphatase (DUSP) family plays an important role in response to adverse external factors. In this study, the DUSP5 from Epinephelus coioides, an important marine fish in Southeast Asia and China, was isolated and characterized. As expected, E. coioides DUSP5 contained four conserved domains: a rhodanese homology domain (RHOD); a dual-specificity phosphatase catalytic domain (DSPc); and two regions of low compositional complexity, indicating that E. coioides DUSP5 belongs to the DUSP family. E. coioides DUSP5 mRNA could be detected in all of the examined tissues, and was mainly distributed in the nucleus. Infection with Singapore grouper iridovirus (SGIV), one of the most important pathogens of marine fish, could inhibit the expression of E. coioides DUSP5. The overexpression of DUSP5 could significantly downregulate the expression of the key SGIV genes (MCP, ICP18, VP19, and LITAF), viral titers, the activity of NF-κB and AP-I, and the expression of pro-inflammatory factors (IL-6, IL-8, and TNF-α) of E. coioides, but could upregulate the expressions of caspase3 and p53, as well as SGIV-induced apoptosis. The results demonstrate that E. coioides DUSP5 could inhibit SGIV infection by regulating E. coioides immune-related factors, indicating that DUSP5 might be involved in viral infection.

Curcumin Alleviates Singapore Grouper Iridovirus-Induced Intestine Injury in Orange-Spotted Grouper (Epinephelus coioides).

Singapore grouper iridovirus (SGIV) is a new ranavirus species in the Iridoviridae family, whose high lethality and rapid spread have resulted in enormous economic losses for the aquaculture industry. Curcumin, a polyphenolic compound, has been proven to possess multiple biological activities, including antibacterial, antioxidant, and antiviral properties. This study was conducted to determine whether curcumin protected orange-spotted grouper (Epinephelus coioides) from SGIV-induced intestinal damage by affecting the inflammatory response, cell apoptosis, oxidative stress, and intestinal microbiota. Random distribution of healthy orange-spotted groupers (8.0 ± 1.0 cm and 9.0 ± 1.0 g) into six experimental groups (each group with 90 groupers): Control, DMSO, curcumin, SGIV, DMSO + SGIV, and curcumin + SGIV. The fish administered gavage received DMSO dilution solution or 640 mg/L curcumin every day for 15 days and then were injected intraperitoneally with SGIV 24 h after the last gavage. When more than half of the groupers in the SGIV group perished, samples from each group were collected for intestinal health evaluation. Our results showed that curcumin significantly alleviated intestine damage and repaired intestinal barrier dysfunction, which was identified by decreased intestine permeability and serum diamine oxidase (DAO) activity and increased expressions of tight junction proteins during SGIV infection. Moreover, curcumin treatment suppressed intestinal cells apoptosis and inflammatory response caused by SGIV and protected intestinal cells from oxidative injury by enhancing the activity of antioxidant enzymes, which was related to the activation of nuclear factor erythroid 2-related factor 2 (Nrf2) signaling. Moreover, we found that curcumin treatment restored the disruption of the intestinal microbiota caused by SGIV infection. Our study provided a theoretical basis for the functional development of curcumin in aquaculture by highlighting the protective effect of curcumin against SGIV-induced intestinal injury.

Protective role of curcumin in Singapore grouper iridovirus-induced liver injury in orange-spotted grouper (Epinephelus coioides)

Curcumin, which is extracted from the traditional Chinese medicine turmeric, is a polyphenolic compound with a variety of biological activities, including immunoenhancement, antioxidant, and antibacterial properties. Singapore grouper iridovirus (SGIV) is a highly contagious viral pathogen that is capable of interfering with the organ function and destroying the immune system of the host, resulting in fish mortality and it has caused significant economic losses to the grouper aquaculture industry. The antiviral activity of curcumin has been demonstrated, but its protective effect against fish virus-induced liver injury requires further investigation. In this study, healthy grouper were infected with SGIV after receiving a curcumin gavage once a day for 15 days and the liver health was assessed. Our results showed that SGIV infection of grouper resulted in severe liver injury as evidenced by histopathological abnormalities, elevated hepatic reactive oxygen species and malondialdehyde, apoptosis and endoplasmic reticulum (ER) stress. On the other hand, we discovered that gavage with curcumin boosted fish immunity and reduced SGIV-induced mortality. Curcumin also ameliorated SGIV-induced liver injury, which might be a result of the modulation of the nuclear factor kappa-B (NF-κB) signaling pathway to reduce liver inflammation, the increase of liver antioxidant levels, the reduction of caspase families-dependent hepatocyte apoptosis, and the relief of liver ER stress by inhibiting heavy-chain binding protein (BIP)/ PKR-like ER kinase (PERK)/ eukaryotic initiation factor 2α (eIF2α)/ activating transcription factor 4 (ATF4) but not inosital-requiring enzyme-1 (IRE1)/ X-box binding protein 1 (XBP1) or ATF6 signaling pathways. This study demonstrated that curcumin possessed strong antiviral activity in fish and highlighted its protective effect on liver of grouper infected with SGIV, providing a theoretical foundation for the functional development of curcumin in aquaculture.

Oral immunizations with Bacillus subtilis spores displaying VP19 protein provide protection against Singapore grouper iridovirus (SGIV) infection in grouper.

Disease caused by Singapore grouper iridovirus (SGIV) results in major economic losses in the global grouper aquaculture industry. Vaccination is considered to be the most effective way to protect grouper from SGIV. In this study, the spores of Bacillus subtilis (B.subtilis) WB600 were utilized as the vehicle that the VP19 protein was displayed on the spores surface. To further investigate the effect of oral vaccination, the grouper were orally immunized with B.s-CotC-19 spores. After challenged, the survival rate of grouper orally vaccinated with B.s-CotC-19 spores was 34.5% and the relative percent survival (RPS) was 28.7% compared to the PBS group. Moreover, the viral load in the tissues of the B.s-CotC-19 group was significantly lower than that of the PBS group. The histopathological sections of head kidney and liver tissue from the B.s-CotC-19 group showed significantly less histopathology compared to the PBS group. In addition, the specific IgM levels in serum in the B.s-CotC-19 group was higher than those in the PBS group. In the hindgut tissue, the immune-related gene expression detected by quantitative real-time PCR (qRT-PCR) exhibited an increasing trend in different degrees in the B.s-CotC-19 group, suggesting that the innate and adaptive immune responses were activated. These results indicated that the oral administration of recombinant B.subtilis spores was effective for preventing SGIV infection. This study provided a feasible strategy for the controlling of fish virus diseases.

Fish SCD1 promotes SGIV infection via modulating the formation of lipid droplets and TBK1/MDA5-activated IFN signal pathway

Stearoyl CoA desaturase (SCD1), the rate-limiting enzyme for monounsaturated fatty acid production, involved in host metabolic regulation. Recent reports indicated that SCD1 played a crucial role in the process of viral infection. To investigate the function of SCD1 in teleost fish and the role it played in fish virus infection, EcSCD1 was cloned and characterized in the orange-spotted grouper, Epinephelus coioides. The open reading frame (ORF) of EcSCD1 consisted of 1005 nucleotides encoding a protein containing 334 amino acids with a predicted molecular weight of 38.33 kDa, which shared 74.05% and 60.48% identity with zebrafish and human, respectively. EcSCD1 possessed four transmembrane domains and three conserved His-rich regions. In healthy groupers, EcSCD1 was primarily expressed in the liver and brain. Subcellular localization assay revealed that EcSCD1 was mainly distributed in the cytoplasm with uniform distribution or dot-like aggregation forms, and EcSCD1 was co-localized with the endoplasmic reticulum (ER). Singapore grouper iridovirus (SGIV) as well as poly (dA: dT) stimulation induced the expression of EcSCD1. On the other hand, EcSCD1 overexpression increased SGIV infection and SCD1 enzymatic product oleic acid (OA) recovered SGIV infection suppressed by SCD1 inhibitor. In addition, EcSCD1 overexpression facilitated the formation of lipid droplets (LDs), which was crucial for SGIV replication. Furthermore, EcSCD1 overexpression down-regulated the expression of interferon signaling molecules and the activities of interferon (IFN)1/3 and interferon stimulated response element (ISRE) promotors. Moreover, EcSCD1 overexpression restricted TANK binding kinase 1 (TBK1)−/melanoma differentiation-associated protein 5 (MDA5)-, but not mitochondrial antiviral signaling protein (MAVS)-evoked interferon immune responses. Overall, our findings offered novel insights into the regulatory mechanism of EcSCD1 during virus infection.

Near-atomic architecture of Singapore grouper iridovirus and implications for giant virus assembly

Singapore grouper iridovirus (SGIV), one of the nucleocytoviricota viruses (NCVs), is a highly pathogenic iridovirid. SGIV infection results in massive economic losses to the aquaculture industry and significantly threatens global biodiversity. In recent years, high morbidity and mortality in aquatic animals have been caused by iridovirid infections worldwide. Effective control and prevention strategies are urgently needed. Here, we present a near-atomic architecture of the SGIV capsid and identify eight types of capsid proteins. The viral inner membrane-integrated anchor protein colocalizes with the endoplasmic reticulum (ER), supporting the hypothesis that the biogenesis of the inner membrane is associated with the ER. Additionally, immunofluorescence assays indicate minor capsid proteins (mCPs) could form various building blocks with major capsid proteins (MCPs) before the formation of a viral factory (VF). These results expand our understanding of the capsid assembly of NCVs and provide more targets for vaccine and drug design to fight iridovirid infections.

Singapore grouper iridovirus infection counteracts poly I:C induced antiviral immune response in vitro

Groupers are important mariculture fish in South China and Southeast Asian countries. However, the increasing frequency of infectious disease outbreaks has caused great economic losses in the grouper industry. Among these pathogens, Singapore grouper iridovirus (SGIV) infection causes high mortality in larval and juvenile stages of grouper. However, the mechanism underlying the action of viral manipulation on cellular immune response still remained largely uncertain. Here, using RNA-seq technology, we investigated the regulatory roles of SGIV infection on synthetic RNA duplex poly I:C induced immune response in vitro. Using reporter gene assays, we found that SGIV infection decreased poly I:C induced interferon promoter activation. Transcriptomic analysis showed that the mRNA expression levels of 2238 genes were up-regulated, while 1247 genes were down-regulated in poly I:C transfected grouper spleen (GS) cells. Interestingly, SGIV infection decreased the expression of 1479 up-regulated genes and increased the expression of 297 down-regulated genes in poly I:C transfected cells. The differentially expressed genes (DEGs) down-regulated by SGIV were directly related to immune, inflammation and viral infection, and JUN, STAT1, NFKB1, MAPK14A, TGFB1 and MX were the 6 top hub genes in the down-regulated DEGs’ protein-protein interaction (PPI) network. Furthermore, quantitative real-time PCR (qPCR) analysis confirmed that the interferon signaling and inflammatory-related genes, including cGAS, STING, TBK1, MAVS, TNF, IRAK4 and NOD2 were up-regulated by poly I:C stimulation, but all significantly down-regulated after SGIV infection. Thus, we speculated that SGIV infection counteracted poly I:C induced antiviral immune response and this ability helped itself to escape host immune surveillance. Together, our data will contribute greatly to understanding the potential immune evasion mechanism of iridovirus infection in vitro.

Antiviral immunity of grouper MAP kinase phosphatase 1 to Singapore grouper iridovirus infection

Singapore grouper iridovirus (SGIV), with various mechanisms for evading and modulating host, has inflicted heavy economic losses in the grouper aquaculture. MAP kinase phosphatase 1 (MKP-1) regulates mitogen-activated protein kinases (MAPKs) to mediate the innate immune response. Here, we cloned EcMKP-1, an MKP-1 homolog from the orange-spotted grouper Epinephelus coioides, and investigated its role in the infection of SGIV. In juvenile grouper, EcMKP-1 was highly upregulated and peaked at different times after injection with lipopolysaccharide, polyriboinosinic polyribocytidylic acid and SGIV. EcMKP-1 expression in heterologous fathead minnow cells was able to suppress SGIV infection and replication. Furthermore, EcMKP-1 was a negative regulator of c-Jun N-terminal kinase (JNK) phosphorylation early in SGIV infection. EcMKP-1 decreased the apoptotic percentage and caspase-3 activity during the late stage of SGIV replication. Our results demonstrate critical functions of EcMKP-1 in antiviral immunity, JNK dephosphorylation and anti-apoptosis during SGIV infection.

Passive immune protection against Singapore grouper iridovirus in groupers by oral feeding of egg yolk antibody

Singapore grouper iridovirus (SGIV) is a major infectious virus in fish, which causes high mortality and considerable economic damage to cultured groupers. However, effective drugs and commercialized vaccines for SGIV are lacking. The main aim of this study was to evaluate the protective effect of immunoglobulin of yolk (IgY) against SGIV infection in groupers. In this study, specific anti-SGIV IgY was produced by immunizing hens with formalin-inactivated SGIV. IgY was extracted and purified from egg yolk using water dilution, two-step salt precipitation, and dialysis. The titers of specific IgY increased significantly after the third immunization, peaking at week 10 until week 12 after the first immunization. Western blotting and indirect immunofluorescence showed that anti-SGIV IgY recognized the SGIV major capsid protein and SGIV virions. In addition, in vitro antibody neutralization assays showed that IgY could effectively neutralize SGIV and significantly inhibit SGIV infection in grouper spleen cells. Before SGIV infection, the groupers were continuously fed with standard feed mixed with specific or nonspecific IgY. The relative protection rate in the specific IgY group was approximately 40% compared with the nonspecific IgY group. Histopathological analysis showed that specific IgY prevented pathological changes in the groupers' liver and head kidney tissues. IgY exerted protective effects probably by reducing the infection of target organ cells and attenuating the inflammatory response, as indicated by the reduced copy numbers of SGIV and the reduced expression levels of tumor necrosis factor-α, cluster of differentiation 8-α, interleukin-8, cluster of differentiation 4–1, interferon-induced protein 35, interferon-γ, and MX protein in the spleen, liver, trunk kidney, and head kidney tissues. These results suggest that anti-SGIV IgY is a promising strategy for preventing SGIV infection in groupers.