Infectious Spleen And Kidney Necrosis Virus Infection Research Articles

Molecular mechanism of infectious spleen and kidney necrosis virus in manipulating the hypoxia-inducible factor pathway to augment virus replication

ABSTRACT Infectious spleen and kidney necrosis virus (ISKNV), a member of the genus Megalocytivirus in the family Iridoviridae, can infect over 50 fish species and cause significant economic losses in Asia. Our previous study showed that hypoxia triggers the hypoxia-inducible factor pathway (HIF-pathway), leading to increased replication of ISKNV through promoting the upregulation of viral hypoxic response genes like orf077r. This study delved into the molecular mechanism of how ISKNV manipulates the HIF-pathway to enhance its replication. In vitro and in vivo experiments confirmed that ISKNV infection activated the HIF-pathway, which in turn promoted ISKNV replication. These findings suggest that ISKNV actively manipulates the HIF-pathway. Co-immunoprecipitation experiments revealed that the ISKNV-encoded protein VP077R interacts with the Von Hippel−Lindau (VHL) protein at the HIF-binding region, competitively inhibiting the interaction of HIF-1α with VHL. This prevents HIF degradation and activates the HIF-pathway. Furthermore, VP077R interacts with factor-inhibiting HIF (FIH), recruiting FIH and S-phase kinase-associated protein 1 (Skp1) to form an FIH – VP077R – Skp1 complex. This complex promotes FIH protein degradation via ubiquitination, further activating the HIF-pathway. These findings indicated that ISKNV takes over the HIF-pathway by releasing two “brakes” on this pathway (VHL and FIH) via VP077R, facilitating virus replication. We speculate that hypoxia initiates a positive feedback loop between ISKNV VP077R and the HIF pathway, leading to the outbreak of ISKNV disease. This work offers valuable insights into the complex interactions between the environment, host, and virus.

Interaction of Teleost Fish TRPV4 with DEAD Box RNA Helicase 1 Regulates Iridovirus Replication.

Viral diseases are a significant risk to the aquaculture industry. Transient receptor potential vanilloid 4 (TRPV4) has been reported to be involved in regulating viral activity in mammals, but its regulatory effect on viruses in teleost fish remains unknown. Here, the role of the TRPV4-DEAD box RNA helicase 1 (DDX1) axis in viral infection was investigated in mandarin fish (Siniperca chuatsi). Our results showed that TRPV4 activation mediates Ca2+ influx and facilitates infectious spleen and kidney necrosis virus (ISKNV) replication, whereas this promotion was nearly eliminated by an M709D mutation in TRPV4, a channel Ca2+ permeability mutant. The concentration of cellular Ca2+ increased during ISKNV infection, and Ca2+ was critical for viral replication. TRPV4 interacted with DDX1, and the interaction was mediated primarily by the N-terminal domain (NTD) of TRPV4 and the C-terminal domain (CTD) of DDX1. This interaction was attenuated by TRPV4 activation, thereby enhancing ISKNV replication. DDX1 could bind to viral mRNAs and facilitate ISKNV replication, which required the ATPase/helicase activity of DDX1. Furthermore, the TRPV4-DDX1 axis was verified to regulate herpes simplex virus 1 replication in mammalian cells. These results suggested that the TRPV4-DDX1 axis plays an important role in viral replication. Our work provides a novel molecular mechanism for host involvement in viral regulation, which would be of benefit for new insights into the prevention and control of aquaculture diseases. IMPORTANCE In 2020, global aquaculture production reached a record of 122.6 million tons, with a total value of $281.5 billion. Meanwhile, frequent outbreaks of viral diseases have occurred in aquaculture, and about 10% of farmed aquatic animal production has been lost to infectious diseases, resulting in more than $10 billion in economic losses every year. Therefore, an understanding of the potential molecular mechanism of how aquatic organisms respond to and regulate viral replication is of great significance. Our study suggested that TRPV4 enables Ca2+ influx and interactions with DDX1 to collectively promote ISKNV replication, providing novel insights into the roles of the TRPV4-DDX1 axis in regulating the proviral effect of DDX1. This advances our understanding of viral disease outbreaks and would be of benefit for studies on preventing aquatic viral diseases.

A New Cell Line Derived from the Caudal Fin of the Dwarf Gourami (Trichogaster lalius) and Its Susceptibility to Fish Viruses.

The detection of megalocytiviruses, especially the infectious spleen and kidney necrosis virus (ISKNV), in ornamental fish has increased with the rapid growth of the ornamental fish industry. In this study, dwarf gourami fin (DGF) cells derived from the caudal fin of the dwarf gourami (Trichogaster lalius), which is highly susceptible to red sea bream iridovirus (RSIV) and ISKNV, were established and characterized. The DGF cells were grown at temperatures ranging from 25 °C to 30 °C in Leibovitz's L-15 medium supplemented with 15% fetal bovine serum and were subcultured for more than 100 passages, predominantly with epithelial-like cells. DGF cells had a diploid chromosome number of 2n = 44. Although the initial purpose of this study was to establish a cell line for the causative agents of red sea bream iridoviral disease (RSIV and ISKNV), DGF cells were also susceptible to rhabdoviruses (viral hemorrhagic septicemia virus, hirame rhabdovirus, and spring viraemia of carp virus), exhibiting a significant cytopathic effect characterized by cell rounding and lysis. Additionally, viral replication and virion morphology were confirmed using virus-specific conventional polymerase chain reaction and transmission electron microscopy. Furthermore, both RSIV and ISKNV were replicated at high concentrations in DGF cells compared to other cell lines. Notably, the DGF cells maintained a monolayer during ISKNV infection, indicating the possibility of persistent infection. Thus, DGF can be used for viral diagnosis and may play a critical role in advancing our understanding of ISKNV pathogenesis.

Partial validation of a TaqMan quantitative polymerase chain reaction for the detection of the three genotypes of Infectious spleen and kidney necrosis virus.

Megalocytiviruses (MCVs) are double-stranded DNA viruses known to infect important freshwater and marine fish species in the aquaculture, food, and ornamental fish industries worldwide. Infectious spleen and kidney necrosis virus (ISKNV) is the type species within the genus Megalocytivirus that causes red seabream iridoviral disease (RSIVD) which is a reportable disease to the World Animal Health Organization (WOAH). To better control the transboundary spread of this virus and support WOAH reporting requirements, we developed and partially validated a TaqMan real-time qPCR assay (ISKNV104R) to detect all three genotypes of ISKNV, including the two genotypes that cause RSIVD. Parameters averaged across 48 experiments used a 10-fold dilution series of linearized plasmid DNA (107-101 copies), carrying a fragment of the three-spot gourami iridovirus (TSGIV) hypothetical protein revealed that the assay was linear over 7 orders of magnitude (107-101), a mean efficiency of 99.97 ± 2.92%, a mean correlation coefficient of 1.000 ± 0.001, and a limit of detection (analytical sensitivity) of ≤10 copies of TSGIV DNA. The diagnostic sensitivity and specificity for the ISKNV104R qPCR assay was evaluated and compared to other published assays using a panel of 397 samples from 21 source populations with different prevalence of ISKNV infection (0-100%). The diagnostic sensitivity and specificity for the ISKNV104R qPCR assay was 91.99% (87.28-95.6; 95% CI) and 89.8% (83.53-94.84). The latent class analysis showed that the ISKNV104R qPCR assay had similar diagnostic sensitivities and specificities with overlapping confidence limits compared to a second TaqMan qPCR assay and a SYBR green assay. This newly developed TaqMan assay represents a partially validated qPCR assay for the detection of the three genotypes of the species ISKNV. The ISKNV104R qPCR assay once fully validated, will serve as an improved diagnostic tool that can be used for ISKNV surveillance efforts and diagnosis in subclinical fish to prevent further spread of MCVs throughout the aquaculture and ornamental fish industries.

Isolation, Characterization, and Transcriptome Analysis of an ISKNV-Like Virus from Largemouth Bass.

Largemouth bass (Micropterus salmoides) is an important commercial fish farmed in China. Challenges related to diseases caused by pathogens, such as iridovirus, have become increasingly serious. In 2017, we detected iridovirus-infected diseased largemouth bass in Zunyi, Guizhou Province. The isolated virus was identified as an infectious spleen and kidney necrosis virus (ISKNV)-like virus (ISKNV-ZY). ISKNV-ZY induces a cytopathic effect after infecting mandarin fish brain (MFB) cells. Abundant hexagonal virus particles were observed in the cytoplasm of ISKNV-ZY-infected MFB cells, using electron microscopy. The whole genome of ISKNV-ZY contained 112,248 bp and 122 open reading frames. Phylogenetic tree analysis showed that ISKNV-ZY was most closely related to BCIV, indicating that it is an ISKNV-like megalocytivirus. ISKNV-ZY-infected largemouth bass started to die on day six and reached a death peak on days 7-8. Cumulative mortality reached 100% on day 10. Using RNA sequencing-based transcriptome analysis after ISKNV-ZY infection, 6254 differentially expressed unigenes (DEGs) were identified, of which 3518 were upregulated and 2673 downregulated. The DEGs were associated with endocytosis, thermogenesis, oxidative phosphorylation, the JAK-STAT signaling pathway, the MAPK signaling pathway, etc. These results contribute to understanding the molecular regulation mechanism of ISKNV infection and provide a basis for ISKNV prevention.

A recombinant baculovirus vector vaccine (BacMCP) against the infectious spleen and kidney necrosis virus (ISKNV).

The infectious spleen and kidney necrosis virus (ISKNV) is a highly lethal virus, which has brought significant losses to aquaculture. Therefore, a new vaccine against ISKNV with high efficiency, safety and convenience must be developed. While baculoviruses are more commonly used as protein expression systems for vaccine antigen production, this paper used baculovirus technology to develop a live-vector vaccine, BacMCP, which contains the coding sequence of the major capsid protein (MCP) (GenBank accession no. AF371960) of ISKNV and is driven by a CMV promoter. Real-time PCR and immunofluorescence showed that the MCP gene was successfully delivered to and expressed in fish cells and tissues inoculated with BacMCP. Immune-related gene (IgM, TGF-β, IL-1, IL-8, TNF-α) expression was induced in BacMCP-treated groups of largemouth bass compared with control groups. Specific antibodies could be detected in the serum of BacMCP injection-vaccinated largemouth bass by ELISA. After injection or immersion vaccination with BacMCP for 21 days, largemouth bass were infected with ISKNV. The immune effect of the injected immunization on fish in different sizes was evaluated. The vaccine efficacy of injection-vaccinated bass was 100% in small bass and 85.7% in large bass. The vaccine efficacy of immersion-vaccinated small bass was 77.3%. This study suggested that BacMCP can be used as a vector-based vaccine candidate to prevent the diseases caused by ISKNV infection.

Infectious spleen and kidney necrosis virus induces the reactive oxidative species/Nrf2-mediated oxidative stress response for the regulation of mitochondrion-mediated Bax/Bak cell death signals in GF-1 cells.

Infectious spleen and kidney necrosis virus (ISKNV) infections can trigger host cell death and are correlated with viral replication; however, they have rarely been considered in terms of the host organelle involvement. In the present study, we demonstrated that ISKNV triggered an oxidative stress signal in the Nrf2-mediated oxidative stress response and induced stress signals for Bax/Bak-mediated host cell death in fish GF-1 cells. The results showed that after ISKNV infection, the levels of reactive oxidative species (ROS) increased by 60–80% from day 3 to day 5, as assessed by an H2DCFDA assay for tracing hydrogen peroxide (H2O2), which was correlated with up to a one-fold change in the fish GF-1 cells. Furthermore, we found that ISKNV infection induced Nrf2-mediated ROS stress signals from D1 to D5, which were correlated with the upregulation of antioxidant enzymes, such as catalase, SOD1, and SOD2; these effects were blocked by the antioxidants GSH and NAC. By analyzing Nrf2-mediated ROS stress signals for cell death regulation via an apoptotic assay, we found that treatment with antioxidants reduced annexin-V-positive signals by 10% (GSH) to 15% (NAC); moreover, necrotic-positive signals were reduced by 6% (GSH) and 32% (NAC) at day 5 (D5) in GF-1 cells, as indicated by PI staining. Furthermore, we found that Nrf2-mediated ROS stress regulated mitochondrion-mediated Bax/Bak death signals at D3 and D5; this was effectively blocked by antioxidant treatment in the GF-1 cells, as demonstrated by a JC1 assay (ΔΨm) and western blot analysis. In addition, we found that downstream signals for caspase-9 and -3 activation were apparently blocked by antioxidant treatment at D3 and D5. Finally, we found that treatment with GSH and NAC reduced major capsid protein (MCP) expression and virus titer (TCID50%) by up to 15-fold at D5 in GF-1 cells. Thus, our data suggest that ISKNV can induce ROS production, which triggers Nrf2-mediated stress signals. Then, these stress signals can regulate mitochondrion-mediated Bax/Bak apoptotic signaling, which is connected to downstream caspase-9 and -3 activation. If ISKNV-induced Nrf2-mediated stress signaling is blocked, then the antioxidants GSH and NAC can also suppress apoptotic signals or reduce viral replication. These findings may provide insights into the control and treatment of double-stranded DNA viruses.

Generation of monoclonal antibodies against heterologously expressed major capsid protein of infectious spleen and kidney necrosis virus (ISKNV)

Infectious spleen and kidney necrosis virus (ISKNV) is one of the detrimental viruses causing economic losses in several fish species including Asian sea bass (Lates calcarifer). In this study, monoclonal antibodies specific to major capsid protein (MCP) of ISKNV were generated. The MCP encoding gene of ISKNV genotype II was cloned into pGEX-6P-1 and transformed into E. coli. After induction, a glutathione-S-transferase (GST)-MCP fusion protein was produced and used for immunization of Swiss mice for monoclonal antibody (MAb) production. Five MAbs specific to MCP were obtained after selection against naturally ISKNV-infected Asian sea bass by dot blotting, Western blotting and immunohistochemistry. All MAbs showed no cross-reaction with fish tissues, fish viruses including tilapia lake virus (TiLV), nervous necrosis virus (NNV), scale drop disease virus (SDDV) and eight bacterial species commonly found in diseased fish. The detection sensitivity of MAbs was inferior to that of one step PCR. However, a combination of two MAbs enhanced the detection limit about two-fold. The intense immunoreactivity staining could be observed in liver, kidney, gill, intestine, stomach, and spleen tissues from ISKNV-infected fish by immunohistochemical analysis. All five MAbs can be used to detect naturally ISKNV-infected fish from two different farms and phylogenetic tree analysis based on MCP gene sequences demonstrated ISKNV genotype I infection in diseased fish. In addition, all MAbs displayed immunoreactivity to red sea bream iridovirus (RSIV) in the GF cell culture fluid by dot blotting. Therefore, the generated MAbs could be used for detection of ISKNV infection by various immunological assays and might be able to detect RSIV infection as well.

Neomycin inhibits Megalocytivirus infection in fish by antagonizing the increase of intracellular reduced glutathione

Infectious spleen and kidney necrosis virus (ISKNV) is the type species of the Megalocytivirus genus that infects a number of marine and freshwater fishes, causing huge economic losses in aquaculture. The ISKNV infection leads to increase of reducing power in cells. As the antibiotic neomycin can promote the production of reactive oxygen species (ROS) in animal cells, in the current study, the potential therapeutic effect of neomycin on ISKNV infection was explored. We showed that neomycin could decrease the reducing power in cultured MFF-1 cells and inhibit ISKNV infection by antagonizing the shift of the cellular redox balance toward reduction. In vivo experiments further demonstrated that neomycin treatment significantly suppresses ISKNV infection in mandarin fish. Expression of the major capsid protein (MCP) and the proportion of infected cells in tissues were down-regulated after neomycin treatment. Furthermore, neomycin showed complex effects on expression of a set of antiviral related genes of the host. Taking together, the current study suggested that the viral-induced redox imbalance in the infected cells could be used as a target for suppressing ISKNV infection. Neomycin can be potentially utilized for therapeutic treatment of Megalocytivirus diseases by antagonizing intracellular redox changes.

Development of two cell lines from yellowfin seabream Acanthopagrus latus fin and brain suitable for propagating SDDV but not for ISKNV, MRV and GIV

Scale drop disease virus (SDDV), a very distinct member of genus Megalocytivirus in family Iridoviridae, was recently confirmed as the most alarming causative agent associated with yellowfin seabream Acanthopagrus latus ascites diseases in South China. To better understand how host-pathogen interacting and developing cell-culture based vaccine, establishment of highly susceptible cell line generating from the natural host fish for SDDV infection will be a top priority. Here, two novel cell lines from yellowfin seabream fin and brain were established and characterized, and designated as YFSBF and YFSBB, respectively. Morphologically, both cell lines predominantly consisted of fibroblast-like cells and grew well in Dulbecco's modified Eagle medium supplemented with 10% fetal bovine serum. Karyotype analysis revealed that chromosome numbers of YFSBF and YFSBB were wildly distributed without obvious featured chromosomic modes. Viral susceptibility tests showed that SDDV could propagate well in both YFSBF and YFSBB, and YFSBF showed more susceptible to SDDV than YFSBB did, being confirmed by cytopathic effect observation, absolute real-time quantitative PCR determination, immunofluorescence assay, Western blot analysis and transmission electron microscopy examination. Viral susceptibility tests also confirmed that SDDV could effectively propagate in MFF-1 cell, a highly susceptible host cell line for infectious spleen and kidney necrosis virus (ISKNV), whereas either YFSBF or YFSBB was refractory to ISKNV infection. Moreover, neither YFSBF nor YFSBB could support the effective propagations of mandarin ranavirus (MRV) and grouper ranavirus (GIV-R). Collectedly, this study provides some important information for better understanding the differential infection profile among SDDV, ISKNV and other fish iridovirus.

Infectious Spleen and Kidney Necrosis Virus (ISKNV) Triggers Mitochondria-Mediated Dynamic Interaction Signals via an Imbalance of Bax/Bak over Bcl-2/Bcl-xL in Fish Cells.

The molecular pathogenesis of infectious spleen and kidney necrosis virus (ISKNV) infections is important but has rarely been studied in connection to host organelle behavior. In the present study, we demonstrated that ISKNV can induce host cell death via a pro-apoptotic Bcl-2 and anti-apoptotic Bcl-2 family member imbalance in mitochondrial membrane potential (MMP or ΔΨm) regulation in GF-1 cells. The results of our study on ISKNV infection showed that it can induce host cell death by up to 80% at day 5 post-infection. Subsequently, in an apoptotic assay, ISKNV infection was seen to induce an increase in Annexin-V-positive signals by 20% and in propidium iodide (PI) staining-positive signals by up to 30% at day 5 (D5) in GF-1 cells. Then, through our studies on the mechanism of cell death in mitochondria function, we found that ISKNV can induce MMP loss by up to 58% and 78% at days 4 and 5 with a JC1 dye staining assay. Furthermore, we found that pro-apoptotic members Bax and Bak were upregulated from the early replication stage (day one) to the late stage (day 5), but the expression profiles were very dynamically different. On the other hand, by Western blotted analysis, the anti-apoptotic members Bcl-2 and Bcl-xL were upregulated very quickly at the same time from day one (two-fold) and continued to maintain this level at day five. Finally, we found that pro-apoptotic death signals strongly activated the downstream signals of caspase-9 and -3. Taken together, these results suggest that ISKNV infection can induce Bax/Bak-mediated cell death signaling downstream of caspase-9 and -3 activation. During the viral replication cycle with the cell death induction process, the anti-apoptotic members Bcl-2/Bcl-xL interacted with the pro-apoptotic members Bax/Bak to maintain the mitochondrial function in the dynamic interaction so as to maintain the MMP in GF-1 cells. These findings may provide insights into DNA-virus control and treatment.

A natural outbreak of infectious spleen and kidney necrosis virus threatens wild pearlspot, Etroplus suratensis in Peechi Dam in the Western Ghats biodiversity hotspot, India.

A large-scale mortality of pearlspot, Etroplus suratensis was reported from Peechi Dam, an artificial tropical lake made for irrigation and drinking water supply in Kerala, India during 2018. This dam is located in the premises of Western Ghats, recognized as one of the biodiversity hotspots of the world. The objective of this study was to identify the aetiological agent of this large-scale mortality of E. suratensis by systematic diagnostic investigation and identification of the pathogen. Virus isolation was carried out on a species-specific pearlspot fin (PSF) cell line. Infected PSF cells showed cytopathic effects (CPEs) like cell shrinkage, rounding, enlargement, clustering, and subsequent detachment of cells with a high viral titre of 106⋅95 TCID50 ml-1 at 8 days post-inoculation (dpi). Histopathological examination of the fish showed the presence of numerous abnormal enlarged basophilic cells and intracytoplasmic eosinophilic inclusions in the liver. Moreover, transmission electron microscopy (TEM) analysis revealed the presence of large numbers of 125-132nm viral particles in the spleen tissues. PCR amplification and phylogenetic analysis of the major capsid protein (MCP) gene sequence confirmed that the causative agent was infectious spleen and kidney necrosis virus (ISKNV) of the genus Megalocytivirus. The experimental infection recorded 86.7 ± 2.7% mortality in the E. suratensis (body weight 11.01 ± 2.7g; body length 8.01 ± 2.23cm) injected with 1×104⋅25 TCID50 ml-1 ISKNV per fish. Our detailed investigation provided definitive diagnosis of ISKNV in the severe mass mortality event in wild E. suratensis in Peechi Dam, India, adding one more species to expanding host range of ISKNV infection. The high mortality rate of ISKNV infection in pearlspot suggests the perilous nature of this disease, particularly among the wild fish population.

The dynamic immune responses of Mandarin fish (Siniperca chuatsi) to ISKNV in early infection based on full-length transcriptome analysis and weighted gene co-expression network analysis

Mandarin fish (Siniperca chuatsi) been seriously harmed by infectious spleen and kidney necrosis virus (ISKNV) in recent years, but the early immune response mechanism of infection is still unknown. Here, we performed RNA sequencing on the spleens of mandarin fish infected with ISKNV at 0, 12, 24, 48, and 72 h post-infection (hpi) using short-read Illumina RNA sequencing and long-read Pacific Biosciences isoform sequencing to generate a full-length transcriptome. The immune responses of mandarin fish infected with ISKNV at the molecular level were characterized by RNA-seq analysis and weighted gene co-expression network analysis (WGCNA). A total of 26,528 full-length transcript sequences were obtained. There were 2,729 (1,680 up-regulated and 1,112 down-regulated), 1,874 (1,136 up-regulated and 738 down-regulated), 2,032 (1,158 up-regulated and 847 down-regulated), and 4,176 (2,233 up-regulated and 1,943 down-regulated) differentially expressed genes (DEGs) in mandarin fish at 12, 24, 48, and 72 hpi, compared with uninfected fish, respectively. A total of four modules of co-expressed DEGs identified by WGCNA were significantly positively correlated to the four time points after infection, respectively. Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis revealed that the immune-related DEGs in all these modules were mainly enriched in Phagosome, Endocytosis, Herpes simplex infection, and Cytokine-cytokine receptor interaction pathways. Further analysis showed that oher signaling pathways, including CAMs, NOD-like receptor and ER protein processing, Intestinal immune network for IgA production, TLR pathway, and Apoptosis significantly enriched in four modules corresponding to 12, 24, 48, and 72 hpi respectively, had specifically participated in the immune response. Hub genes identified based on the high-degree nodes in the WGCN, including CAM3, IL-8, CCL21, STING, SNX1, PFR and TBK1, and some DEGs such as MHCI, MHCII, TfR, STING, TNF α, TBK1, IRF1, and NF-kB, BCR, IgA and Bcl-XL had involved in dynamic molecular response of mandarin fish to ISKNV infection. In sum, this study provides a set of full-length transcriptome of the spleen tissue of mandarin fish for the first time and revealed a group of immune genes and pathways involved in different temporal responses to ISKNV infection, which has implications for resource conservation and aiding the development of strategies to prevent virus early infection for mandarin fish.

PI3K/AKT/p53 pathway inhibits infectious spleen and kidney necrosis virus infection by regulating autophagy and immune responses

The PI3K/AKT/p53 signaling pathway is activated by various types of cellular stimuli or pathogenic infection, and then regulates fundamental cellular functions to combat these stimulations. Here, we studied the meaningful roles of PI3K/AKT/p53 in regulating cellular machine such as autophagy, immune responses, as well as antiviral activity in Chinese perch brain (CPB) cells infected by infectious spleen and kidney necrosis virus (ISKNV), which is an agent caused devastating losses in mandarin fish (Siniperca chuatsi) industry. We found that ISKNV infection induced up-regulation of host PI3K/AKT/p53 axis, but inhibited autophagy in CPB cells. Interestingly, activation of PI3K/AKT/p53 axis factors trough agonists or overexpression dramatically decreased host autophagy level, inhibited ISKNV replication, and elevated the expression of immune-related genes in CPB cells. In contrast, suppression of PI3K/AKT/p53 pathway by inhibitors or small interfering RNA (siRNA)-mediated gene silence increased the autophagy and ISKNV replication, but down-regulated immune responses in CPB cells. All these results indicate that PI3K/AKT/p53 pathway plays an important role in anti-ISKNV infection and can be used as a new target for controlling ISKNV disease.

MicroRNA expression and analysis of immune-related putative target genes in ISKNV-infected spleen of mandarin fish (Siniperca chuatsi)

Infectious spleen and kidney necrosis virus (ISKNV), a highly infectious and lethal pathogen of mandarin fish (Siniperca chuatsi), has resulted in major aquaculture losses over the past few decades. A lack of understanding of the regulatory mechanism of viral infection has impeded the development of effective measures to control ISKNV infection. MicroRNAs (miRNAs) are a class of non-coding RNAs that regulate the processes of viral infection and are involved in various physiological processes in vivo. In this study, miRNAs involved in the response of mandarin fish to ISKNV infection were identified at several time points following infection through Illumina high-throughput sequencing. A total of 434 (27 known and 407 novel) miRNAs were identified in mandarin fish; 26 (26 up-regulated), 38 (30 up-regulated, 8 down-regulated), and 18 (14 up-regulated and 4 down-regulated) differentially expressed miRNAs (DEMs) were identified at 12, 24, and 48 h post-infection (hpi), respectively. The putative target genes of DEMs were all significantly enriched in host immune response or signaling pathways, such as autophagy, endocytosis, phosphatidylinositol signaling system, Salmonella infection, cell cycle, protein export, and protein processing in endoplasmic reticulum pathway at 12 and 24 hpi and in vascular endothelial growth factor, phosphatidylinositol signaling system and lysosome at 48 hpi. Further analysis indicated that the targeted mRNAs of miRNAs were involved in innate immunity; autophagy and endocytosis might play an important role in immune regulation within 24 hpi with ISKNV. Although the number of DEMs was lowest at 48 hpi, they might still be involved in immunity through the above pathway. We also constructed a miRNA–mRNA targeting network. Generally, the findings of this study provide novel information that advance our understanding of the regulatory mechanisms of miRNA during ISKNV infection in mandarin fish as well as the development of new approaches for preventing ISKNV infection.

Molecular evidence for homologous strains of infectious spleen and kidney necrosis virus (ISKNV) genotype I infecting inland freshwater cultured Asian sea bass (Lates calcarifer) in Thailand.

Infectious spleen and kidney necrosis virus (ISKNV) is a fish-pathogenic virus belonging to the genus Megalocytivirus of the family Iridoviridae. In 2018, disease occurrences (40-50% cumulative mortality) associated with ISKNV infection were reported in grown-out Asian sea bass (Lates calcarifer) cultured in an inland freshwater system in Thailand. Clinical samples were collected from seven distinct farms located in the eastern and central regions of Thailand. The moribund fish showed various abnormal signs, including lethargy, pale gills, darkened body, and skin hemorrhage, while hypertrophied basophilic cells were observed microscopically in gill, liver, and kidney tissue. ISKNV infection was confirmed on six out of seven farms using virus-specific semi-nested PCR. The MCP and ATPase genes showed 100% sequence identity among the virus isolates, and the virus was found to belong to the ISKNV genotype I clade. Koch's postulates were later confirmed by challenge assay, and the mortality of the experimentally infected fish at 21 days post-challenge was 50-90%, depending on the challenge dose. The complete genome of two ISKNV isolates, namely KU1 and KU2, was recovered directly from the infected specimens using a shotgun metagenomics approach. The genome length of ISKNV KU1 and KU2 was 111,487 and 111,610 bp, respectively. In comparison to closely related ISKNV strains, KU1 and KU2 contained nine unique genes, including a caspase-recruitment-domain-containing protein that is potentially involved in inhibition of apoptosis. Collectively, this study indicated that inland cultured Asian sea bass are infected by homologous ISKNV strains. This indicates that ISKNV genotype I should be prioritized for future vaccine research.

Transcriptomic and proteomic analyses of the immune mechanism in pathogenetic and resistant mandarin fish (Siniperca chuatsi) infected with ISKNV

Mandarin fish (Siniperca chuatsi) is one of the most economically important aquaculture species in China. Mandarin fish aquaculture has experienced major losses in the past few decades because of outbreaks of infectious spleen and kidney necrosis virus (ISKNV). Little is known about the immune mechanism of pathogenetic and resistant mandarin fish infected with ISKNV. Here, the transcriptome and proteome of the spleen of mandarin fish on the 8th day after ISKNV infection were analyzed using Illumina NovaSeq 6000 and isobaric tag for relative and absolute quantitation. Histology was observed by staining with hematoxylin and eosin. ISKNV loads were markedly higher in pathogenetic fish than in resistant fish. Serious tissue injury and inflammation were observed in the spleen tissue of pathogenetic fish. A total of 4667 and 3417 differentially expressed genes (DEGs), 258 and 54 differentially expressed proteins (DEPs), and 45 and 7 cor-DEGs-DEPs (i.e., correlation differentially expressed genes and proteins) were identified in pathogenetic fish and resistant fish, respectively. The DEGs were specifically enriched in the FcεRI signaling pathway in resistant fish; the up-regulated genes Rac (Rac1), FcεRIγ, TNFα, and IgE and the down-regulated genes 5-LO and SHIP might play a positive immune-regulatory role in anti-inflammation, resistance to pathogen infection, cytokine expression, and the generation of immune cells. In pathogenetic fish, DEGs and DEPs were specifically enriched in the natural killer cell-mediated cytotoxicity pathway, and the up-regulated genes Fyn and Shc might exacerbate tissue inflammation and inhibit NK cell-mediated cytotoxic pathways; the down-regulated protein perforin and up-regulated protein granzyme might inhibit the penetration of effector proteins and thus their ability to mediate the programmed cell death of ISKNV-infected target cells. The up-regulated cor-DEGs-DEPs BF, C8, and PLG were specifically enriched in the complement and coagulation cascade pathway, and the down-regulated cor-DEGs-DEPs p120ctn and MLC were specifically enriched in the leukocyte transepithelial migration pathway, which might inhibit complement cascade function, accelerate viral infection, and result in an excessive inflammatory effect. The findings of this study provide novel information relating to the molecular mechanism of pathogenicity and the resistance of mandarin fish infected with ISKNV.

Characterization of mandarin fish (Siniperca chuatsi) IL-6 and IL-6 signal transducer and the association between their SNPs and resistance to ISKNV disease

In fish, interleukin-6 (IL-6) is a very important immune-regulatory cytokine that plays a polyfunctional role in inflammation, metabolism, regeneration, and neural processes. IL-6 signal transducer (IL-6ST) is a specific receptor for IL-6 and expressed mainly in immune cells and hepatocytes. In this study, the complete cDNA and genomic DNA sequences of mandarin fish (Siniperca chuatsi) IL-6 and IL-6ST genes were identified and analyzed. Quantitative real-time PCR showed that IL-6 and IL-6ST were chiefly expressed in the immune organs. After challenge with infectious spleen and kidney necrosis virus (ISKNV), the expression levels of IL-6 were significantly up-regulated after 6 h and 24 h in the head kidney and spleen, respectively (p < 0.01), the peak value for both reached at 72 h, IL-6ST increased significantly after 120 h with a peak at 168 h in the head kidney (p < 0.01) and improved markedly at 168 h in the spleen (p < 0.01). Besides, IL-6 and IL-6ST have been identified 3 and 8 single nucleotide polymorphisms (SNPs), respectively. Statistical analysis showed that one SNP locus (1625C/T) in the coding region of IL-6 was significantly related to the resistance of mandarin fish against ISKNV. The 1625C→T locus in the coding region of IL-6 is a synonymous mutation; compared with the susceptible group, the frequency of allele T in the disease resistance group was significantly higher, which may be due to the rare codon produced by the mutation affecting translation. The involvement of IL-6 and IL-6ST in response to ISKNV infection in mandarin fish clearly indicate that the role of SNP markers in IL-6 was associated with the ISKNV resistance, which was demonstrated for the first time in our results. Thus, the current study may provide fundamental information for further breeding of mandarin fish with resistance to ISKNV infection.

Identification of infectious spleen and kidney necrosis virus (ISKNV)-encoded microRNAs.

MicroRNAs (miRNAs) are small noncoding RNAs that post-transcriptionally regulate gene expression by complementary binding to target mRNAs. Virus-encoded miRNAs play important roles in virus life cycle and virus-host interactions. Viruses from the Megalocytivirus genus, family Iridoviridae, infect a wide range of fishes, bringing great challenges to aquaculture. Infectious spleen and kidney necrosis virus (ISKNV) is the type species of the Megalocytivirus genus. In this study, using Illumina sequencing coupled with miRNA precursor prediction and stem-loop real-time PCR, 14 putative ISKNV-encoded miRNAs were preliminarily identified from ISKNV-infected mandarin fish MFF-1 cells. To initially study their functions, inhibitors of the 14 viral miRNAs were synthesized and transfected into MFF-1 cells, which were further infected with ISKNV. The results showed that these viral miRNAs could affect the virus titers in the supernatant of ISKNV-infected cells and the expression of major capsid protein (MCP). Moreover, we observed that inhibition of several ISKNV miRNAs had different effects on MCP expression and on titer of released virus, suggesting complex roles of viral miRNAs in ISKNV infection. The current study may provide a fundamental information for further identification and functional studies on miRNAs encoded by Megalocytivirus.

Transcriptional programs of infectious spleen and kidney necrosis virus (ISKNV) in vitro and in vivo.

Infectious spleen and kidney necrosis virus (ISKNV), causing serious infectious diseases to marine and freshwater fishes, is the type species of the genus Megalocytivirus, family Iridoviridae. In this study, the transcriptional programs of ISKNV in vitro (MFF-1 cells) and in vivo (spleens from mandarin fish) were investigated using real-time PCR. Transcription of all the putative open reading frames (ORFs) of ISKNV was verified. The temporal expression patterns of ISKNV ORFs in vitro and in vivo, including peak expression times (PETs) and relative maximal expression levels, were determined and compared. The K-means clustering with Spearman rank correlation was generated in heat maps constructed based on ISKNV ORF expression profiles in vivo and in vitro. The current study may provide a global picture of ISKNV infection at the transcription level and help better understand the molecular pathogenic mechanism of megalocytiviruses.