Grouper Iridovirus Research Articles

Singapore grouper iridovirus VP12 evades the host antiviral immune response by targeting the cGAS-STING signalling pathway.

The emergence of Singapore grouper iridovirus (SGIV) has caused huge losses to grouper farming. SGIV is a DNA virus and belongs to the genus Ranavirus. Groupers infected with SGIV showed haemorrhaging and swelling of the spleen, with a mortality rate of more than 90% within a week. Therefore, it is of great significance to study the escape mechanism of SGIV from host innate immunity for the prevention and treatment of viral diseases in grouper. In this study, the viral proteins that interact with EccGAS were identified by mass spectrometry, and the SGIV VP12 protein that inhibits cyclic GMP-AMP synthase (cGAS)-stimulator of interferon genes (STING)-mediated antiviral innate immunity was screened by the dual-luciferase reporter gene assay. VP12 belongs to the late gene of the virus. The immunofluorescence analysis demonstrated that VP12 was aggregated and distributed in the cytoplasm during the early stage of virus infection and translocated into the nucleus at the late stage of virus infection. VP12 inhibited the activation of IFN3, ISRE and NF-κB promoter activities mediated by cGAS-STING, EcTBK1 and EcIRF3. Quantitative real-time PCR analysis showed that VP12 inhibited the expression of interferon-related genes, including those mediated by cGAS-STING. VP12 enhanced the inhibition of IFN3, ISRE and NF-κB promoter activity by EccGAS, EccGAS-mab-21 and EccGAS-delete-mab21. The interaction between VP12 and EccGAS was found to be domain independent. The immunoprecipitation results demonstrated that VP12 interacted and co-localized with EccGAS, EcTBK1 and EcIRF3. VP12 degraded the protein levels of EcTBK1 and EcIRF3 and degraded EcIRF3 through the protease pathway. These results suggest that SGIV VP12 protein escapes the cGAS-STING signalling pathway and degrades EcIRF3 protein expression through the protease pathway.

8627 Viral Hormones: The Role Of Viral Insulin/IGF-1 Like Peptides In Host-Pathogen Interactions

Abstract Disclosure: A. Chuard: None. Viruses, encode proteins that are similar to host proteins to manipulate the host. Until our recent discovery of viral hormones, examples of viral mimicry were limited to immunomodulatory proteins and growth factors. We recently showed that six viruses in Iridoviridae family encode genes mimicking human insulin and IGF-1. We previously chemically synthesizing these viral insulin/IGF-1 like peptides (VILPs) and showed that VILPs can bind to human insulin and IGF-1 receptor and further stimulate post-receptor signaling. VILPs also stimulate glucose uptake and proliferation in mammalian cells and lower the blood glucose in mice. Originally, VILP-carrying viruses were isolated from fish; however, the role of VILPs in viral pathogenesis is not studied in depth. Here, we examined the effects of Grouper Iridovirus (GIV, one of the VILP carrying viruses) infection and GIV-VILP on host insulin/IGF signaling system using fish cells.To this end, we chemically synthesized GIV-VILP in its single chain (sc, IGF-1 like) and double chain (dc, insulin-like) forms. Stimulation of grouper kidney (GK) and AB9 zebrafish cells with these peptides, insulin and IGF-1, showed that IGF-1 was the most potent ligand in these cells indicating an abundance of IGF-1 receptor (IGF1R). Further, both forms of the VILPs were as potent as insulin in its activity on receptor phosphorylation and post-receptor signaling. Examining the viral kinetics, we also showed that GIV32 gene (encoding GIV VILP) is an early viral gene in both cell types. Virion analysis through mass spectrometry indicated that VILPs are not viral structural proteins. On the other hand, analysis of the supernatant from infected cells indicated that VILPs are secreted peptides during the viral cycle. Our results with supernatant transfer experiments from infected cells to uninfected cells support the same conclusion that VILPs are secreted. The secreted VILPs initiate autophosphorylation of IGF1R and/or insulin receptor (IR), consequently triggering downstream cellular signaling and metabolism. Through the use of specific inhibitors for each receptor, we illustrated that IR signaling is crucial for GIV viral replication. Surprisingly, inhibition of IGF1R increased viral replication and we are currently examining potential inhibitory effects of GIV VILP. Furthermore, VILPs appear to exert a biased effect on the AKT pathway compared to the Erk pathway. Furthermore, the inhibition of the AKT pathway significantly reduces GIV viral replication. In summary, our findings reveal that GIV-VILP is actively produced during infection and contributes to the viral replication. To our knowledge, VILPs are the first hormone-mimics characterized in viral replication. Elucidating the role of VILPs in host-pathogen interactions, we propose a novel viral pathogenesis mechanism where viruses mimic host hormones to manipulate the host's endocrine system. Presentation: 6/3/2024

Isolation, Identification and Genomic Analysis of Orange-Spotted Grouper Iridovirus Hainan Strain in China.

The orange-spotted grouper (Epinephelus coioides) is an important mariculture fish in China. However, in recent years, with the rapid development of aquaculture activities, outbreaks of viral diseases have affected the grouper aquaculture industry, causing severe economic losses. In the present study, we isolated and identified a virus from diseased, orange-spotted groupers from an aquaculture farm in Hainan Province, China. The isolated virus was identified as orange-spotted grouper iridovirus, hence named the orange-spotted grouper iridovirus Hainan strain (OSGIV-HN-2018-001). OSGIV-HN-2018-001 induces a cytopathic effect after the infection of mandarin fish (Siniperca chuatsi) brain clonal passage (SBC) cells. In addition, the cytoplasm of the OSGIV-HN-2018-001-infected SBC cells was found to contain a large number of hexagonal virus particles with a diameter of approximately 134 nm. Using the Illumina NovaSeq system, we assembled the sequence data and annotated the complete genome of OSGIV-HN-2018-001 (GenBank accession number: PP974677), which consisted of 110,699 bp and contained 122 open reading frames (ORFs). Phylogenetic tree analysis showed that OSGIV-HN-2018-001 was most closely related to ISKNV-ASB-23. The cumulative mortality rate of groupers infected with OSGIV-HN-2018-001 reached 100% on day 8. The spleens were enlarged and blackened after the dissection of the dying groupers. These results contribute to the understanding of the molecular regulatory mechanism of the iridovirus infection and provide a basis for iridovirus prevention.

Multi-epitope Vaccine Design against Grouper Iridovirus (GIV) Using Immuno-bioinformatics Approach

Grouper Iridovirus (GIV) infection induced cell death in grouper spleen cells and caused serious systemic diseases with more than 90% mortality. Therefore, effective strategies are critically needed to prevent economic losses and maintain the sustainability of grouper aquaculture. Using immuno-bioinformatics, this study aimed to create a multi-epitope vaccine (MEV) that would be effective against GIV. The GIV major capsid protein sequences were retrieved from the NCBI proteome database. Out of 284 epitopes, 17 CTL, 12 HTL, and 10 B-cell epitopes were predicted to be antigenic, non-allergenic, and non-toxic. 10 highly antigenic and overlapping epitopes were shortlisted. To generate full-length epitope vaccine candidates, the selected antigenic epitopes were fused with linkers and adjuvants. Four sets of different linker combinations (no linker, GGS, EAAK, GGGS, GPGPG, KK, and AAY) were tested and compared for their antigenicity, allergenicity, and toxicity using several servers. Molecular dynamics simulations with GROMACS were used on the modelled 3D structures to examine their stability. The results of vaccine candidate sequences screening and MD simulation predicted that the structure with GGS linker is relatively stable with a high antigenic index, non-allergenic, and non-toxic. The designed MEV in the present study could be a potential candidate for further vaccine production process against GIV.

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.

Utilization of recombinant Chlorella expressing dsRNA for protection against grouper nervous necrosis virus

In mammals and fish, dsRNA can induce the type I interferon (IFN-I), leading to the expression of antiviral and immune-related genes. The use of microalgae expressing dsRNA as an oral delivery system to activate innate immunity in fish has not been documented. In this study, wild Chlorella sorokiniana was transformed with NNV (nervous necrosis virus) RdRp and GIV (grouper iridovirus) ORF086R dsRNA expression vectors. The recombinant Chlorella strains effectively protected pearl gentian groupers (Epinephelus fuscoguttatus♀ × Epinephelus lanceolatus♂) against NNV and reduced viral titers. The survival rates of the NNV RdRp and GIV ORF086R dsRNA treatment groups increased by 32 % and 38 %, respectively, compared with that of the control group. These findings indicate that recombinant Chlorella has the potential to be used as an oral delivery strategy for protection against NNV disease in groupers.

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.

The Establishment of the Multi-Visual Loop-Mediated Isothermal Amplification Method for the Rapid Detection of Vibrio harveyi, Vibrio parahaemolyticus, and Singapore grouper iridovirus

Groupers are valuable economic fish in the southern sea area of China, but the threat of disease is becoming more and more serious. Vibrio harveyi, V. parahaemolyticus, and Singapore grouper iridovirus (SGIV) are three important pathogens that cause disease in groupers, and infection with either a single one or a mix of these pathogens poses a serious threat to the healthy development of grouper culture. To enhance the rapid diagnosis and screening in the early stages, it is necessary to develop rapid detection methods of these pathogens. To simultaneously and rapidly detect the three pathogens, in this study, we utilized the TolC of V. harveyi, DNAJ of V. parahaemolyticus, and RAD2 of SGIV as the target genes and established a triple visual loop-mediated isothermal amplification (LAMP) method. This LAMP method showed a detection time as fast as 30 min and a high sensitivity of 100 fg/μL. Moreover, this method exhibited strong specificity and no cross-reaction with seven types of Vibrio and Staphylococcus aureus, as well as five common viruses in aquatic animals. Then, the LAMP products were enzymically cut, and three characteristic strips were used to identify the pathogen species. The results of the clinical trials demonstrated that the method could accurately and specifically detect V. harveyi, V. parahaemolyticus, and SGIV in grouper tissues. In summary, this study successfully established a triple visual LAMP rapid detection method for V. harveyi, V. parahaemolyticus, and SGIV. The method offers several advantages including simple equipment, easy operation, rapid reaction, high specificity, high sensitivity, and visual results. It is suitable for the early and rapid diagnosis of groupers infected with V. harveyi, V. parahaemolyticus, and SGIV, thereby providing useful technical support for further application in the large-scale disease surveillance of aquaculture animals.

Singapore grouper iridovirus VP146 modulates the cGAS-STING signaling pathway to escape the interferon immune response

Singapore grouper iridovirus (SGIV) is a large double-stranded DNA virus that has caused significant economic losses to the grouper aquaculture industry. So far, the structure and function of SGIV proteins have been successively reported. In the present paper, the protein of SGIV VP146 was cloned and identified. VP146 was whole-cell distributed in GS cells. VP146 promoted SGIV replication and inhibited the transcription of interferon-related genes as well as pro-inflammatory cytokines in GS cells. In addition, VP146 was involved in the regulation of the cGAS-STING signaling pathway, and decreased cGAS-STING induced the promoter of ISRE and NF-κB. VP146 interacted with the proteins of cGAS, STING, TBK1, and IRF3 from grouper, but did not affect the binding of grouper STING to grouper TBK1 and grouper IRF3. Interestingly, grouper STING was able to affect the intracellular localization of VP146. Four segment structural domains of grouper STING were constructed, and grouper STING-CTT could affect the intracellular localization of VP146. VP146 had no effect on the self-binding of EcSITNG, nor on the binding of EcSTING to EcTBK1 and EcIRF3. Together, the results demonstrated that SGIV VP146 modulated the cGAS-STING signaling pathway to escape the interferon immune response.

SGIV VP82 inhibits the interferon response by degradation of IRF3 and IRF7

During virus-host co-evolution, viruses have developed multiple strategies to dampen IFN response and prevent its antiviral activity in host cells. To date, the interactions between host IFN response and the immune evasion strategies exploited by fish iridoviruses still remain largely uncertain. Here, a potential immune evasion protein candidate of Singapore grouper iridovirus (SGIV), VP82 (encoded by SGIV ORF82) was screened and its roles during viral replication were investigated in detail. Firstly, VP82 overexpression dramatically decreased IFN or ISRE promoter activity and the transcription levels of IFN stimulated genes (ISGs) stimulated by grouper cyclic GMP-AMP synthase (EccGAS)/stimulator of interferon genes (EcSTING), TANK-binding kinase 1 (EcTBK1), IFN regulatory factor 3 (EcIRF3)and EcIRF7. Secondly, Co-IP assays indicated that VP82 interacted with EcIRF3 and EcIRF7, but not EcSTING and EcTBK1, which was consistent with the co-localization between VP82 and EcIRF3 or EcIRF7. Furthermore, VP82 promoted the degradation of EcIRF3 and EcIRF7 in a dose-dependent manner via the autophagy pathway. Finally, VP82 overexpression accelerated SGIV replication, evidenced by the increased transcriptions of viral core genes and viral production. Moreover, the antiviral action of EcIRF3 or EcIRF7 was significantly depressed in VP82 overexpressed cells. Together, VP82 was speculated to exert crucial roles for SGIV replication by inhibiting the IFN response via the degradation of IRF3 and IRF7. Our findings provided new insights into understanding the immune evasion strategies utilized by fish iridovirus through IFN regulation.

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.

Dual-specificity phosphatase 1 inhibits Singapore grouper iridovirus replication via regulating apoptosis in Epinephelus coioides

The dual-specificity phosphatase (DUSP) family plays key roles in the maintenance of cellular homeostasis and apoptosis etc. In this study, the DUSP member DUSP1 of Epinephelus coioides was characterized: the length was 2371 bp including 281 bp 5′ UTR, 911 bp 3′ UTR, and a 1125 bp open reading frame encoding 374 amino acids. E. coioides DUSP1 has two conserved domains, a ROHD and DSPc along with a p38 MAPK phosphorylation site, localized at Ser308. E. coioides DUSP1 mRNA can be detected in all of the tissues examined, and the subcellular localization showed that DUSP1 was mainly distributed in the nucleus. Singapore grouper iridovirus (SGIV) infection could induce the differential expression of E. coioides DUSP1. Overexpression of DUSP1 could inhibit SGIV-induced cytopathic effect (CPE), the expressions of SGIV key genes, and the viral titers. Overexpression of DUSP1 could also regulate SGIV-induced apoptosis, and the expression of apoptosis-related factor caspase 3. The results would be helpful to further study the role of DUSP1 in viral infection.

Polysaccharides derived from Spirulina platensis inhibited Singapore grouper iridovirus by impeding the entry of viral particles

Attributable to the rapid dissemination and high lethality of Singapore grouper iridovirus (SGIV), it has caused significant economic losses for marine fish aquaculture in China and Southeast Asian nations. Hence, there is an urgent need to find antiviral drugs that are both safe and effective. In this study, a novel heteropolysaccharide named Spirulina platensis polysaccharides (SPP) was purified and characterized from S. platensis. The molecular weight of SPP is 276 kDa and it mainly consists of Glc and Rha, followed by minor components such as Gal, Xyl, and Fuc. The backbone of SPP was determined to be →2) –β–Rhap–(1 → 4) –α–Fucp–(1 → [2) –α–Rhap–(1] 2[→6)–α–Glcp–(1] 4[→ 4) –α–Glcp–(1] 8[→ 4) –β–Glcp–(1]2→, with branches of β–Galp, α–Xylp and α–Glcp. SPP significantly inhibited SGIV-induced cytopathic effects (CPEs), viral gene replication and viral protein expression. The antiviral mechanism of SPP was associated with the disruption of SGIV entry to host cells. Furthermore, it was not observed that SPP made statistically significant impact on the expression of interferon-related cytokines. Our results offered novel insights into the potential utilization of spirulina polysaccharides for combating aquatic animal viruses.

Dissecting the early and late endosomal pathways of Singapore grouper iridovirus by single-particle tracking in living cells

Iridoviruses are large DNA viruses that infect a wide range of invertebrates and lower vertebrates, causing serious threats to ecological security and aquaculture industry worldwide. However, the mechanisms underlying intracellular transport of iridovirus remain unknown. In this study, the transport of Singapore grouper iridovirus (SGIV) in early endosomes (EEs) and late endosomes (LEs) was explored by single-particle tracking technology. SGIV employs EEs to move rapidly from the cell membrane to the nucleus, and this long-range transport is divided into “slow-fast-slow” stages. SGIV within LEs mainly underwent oscillatory movements near the nucleus. Furthermore, SGIV entered newly formed EEs and LEs, respectively, possibly based on the interaction between the viral major capsid protein and Rab5/Rab7. Importantly, interruption of EEs and LEs by the dominant negative mutants of Rab5 and Rab7 significantly inhibited the movement of SGIV, suggesting the important roles of Rab5 and Rab7 in virus transport. In addition, it seems that SGIV needs to enter clathrin-coated vesicles to move from actin to microtubules before EEs carry the virus moving along microtubules. Together, our results for the first time provide a model whereby iridovirus transport depending on EEs and LEs, helping to clarify the mechanism underlying iridovirus infection, and provide a convenient tactic to investigate the dynamic infection of large DNA virus.

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.