MFF-1 Cells Research Articles

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.

Deleting ORF71L of infectious spleen and kidney necrosis virus (ISKNV) resulted in virulence attenuation in Mandarin fish

Infectious spleen and kidney necrosis virus (ISKNV), the type species of the genus Megalocytivirus, infects a variety of teleost fish species and causes substantial losses in the aquaculture industry worldwide. ISKNV ORF71L is 1611 bp in length, encodes a 537-amino-acid peptide and was previously identified as a viral structural protein in the ISKNV virion. In this study, the ORF71L deletion mutant virus strain ISKNV-Δ71 was obtained through a homologous recombination approach. The multistep growth curves showed that ISKNV-Δ71 replication was faster than ISKNV-WT replication in mandarin fish fry cells (MFF-1 cells) before 48 h post-infection (hpi). The cumulative mortality of ISKNV-Δ71-infected mandarin fish (Siniperca chuatsi) was lower than that of fish infected with ISKNV-WT. The copy numbers of viral genome equivalents (GEs) in ISKNV-Δ71-infected mandarin fish spleens were also lower than those in ISKNV-WT-infected spleens. Deletion of ORF71L resulted in ISKNV virulence attenuation in mandarin fish. Furthermore, we found that the number of melanomacrophage centers (MMCs) in ISKNV-Δ71-infected mandarin fish spleens was higher than that in ISKNV-WT-infected mandarin fish spleens. Transcriptomic analysis showed that the cytokine–cytokine receptor interaction pathway had the most significant change between ISKNV-Δ71- and ISKNV-WT-infected MFF-1 cells. These results indicated ORF71L is a virulence-related gene of ISKNV. ORF71L could be considered as a potential target for the development of engineered attenuated live vaccines via multigene deletion or as a potential insertion site for exogenous protein expression.

Pathogenicity and histopathology of infectious spleen and kidney necrosis virus genotype II (ISKNV-II) recovering from mass mortality of farmed Asian seabass, Lates calcarifer, in Southern China

Megalocytivirus of family Iridoviridae was considered as one of the most alarming causative agents which caused fatally systemic infection and lead to mass mortalities to world-widely distributed freshwater and marine fish. In recent years, severe diseases with typical symptoms of body blackened, swims slowly to the water surface or surrounding the ponds were observed frequently in cultured Asian seabass, Lates calcarifer, in Zhuhai city of Guangdong province, where is the most important area for Asian seabass culture in mainland China. The causative agents were highly suspected as megalocytiviruses, however, the detailed and systemic studies about these diseases are still lacking. In this study, two cases of high morbidity and mortality of Asian seabass were investigated by systemic isolation and identification of pathogen. Virus isolation by MFF-1 cells and molecular detection confirmed that the causative agents were ISKNV genotype-II (ISKNV-II) of megalocytivirus. The mortalities from natural and artificial infected fish were 85.89% and 83.30%, respectively. A total of 21 single-nucleotide differences, including 20 same-sense mutations, were observed on the ISKNV-II compared with that of ISKNV-I. Histopathology examination showed that numerous abnormal enlarged cells, the most well-known ISKNV-specific clinicopathological features, were observed in spleen, kidney and liver. Results of immunohistochemistry (IHC) and immunofluorescence assay (IFA) examination confirmed that numerous abnormal enlarged cells with abundant viruses existed in these tissues. Moreover, large number of viral particles of diameter 150 nm were also observed both in infected tissues and MFF-1 cells by transmission electron microscope (TEM). Taken together, this is the first study with detailed and robust evidence confirming that ISKNV-II is the causative agent of the severe diseases in farmed Asian seabass in mainland China. This study contributes to the pathogenic and epidemiological studies of Asian seabass iridovirus disease in mainland China and other Asian countries and regions.

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.

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.

The roles of mandarin fish STING in innate immune defense against Infectious spleen and kidney necrosis virus infections

The mandarin fish Siniperca chuatsi is a cultured freshwater fish species that is popular in China because of its high market value. With the development of high-density cultural mode in mandarin fish, viral diseases such as Infectious spleen and kidney necrosis virus (ISKNV) are becoming increasingly serious. Stimulator of interferon genes (STING) is a central component in the innate immune response to cytosolic DNA and RNA derived from different pathogens. However, the roles of STING in innate immune response of mandarin fish remain unknown. In the present study, S. chuatsi STING (scSTING)-mediated host immune response against ISKNV infection was investigated. ScSTING transcription level increased remarkably in response to ISKNV infection, LPS, PMA, or poly (I:C) stimulation in mandarin fish fry (MFF-1) cells. Immunofluorescence results showed that scSTING localized majorly in the endoplasmic reticulum. scSTING overexpression remarkably increased the expression levels of scIFN-h, scMx, scISG15, scPKR, scViperin, scIL-1β, scIL-18, and scTNF-α genes. IFN-β-luciferase report assay results showed that the relative expressions of luciferin were remarkably increased in MFF-1 cells. Site mutation of serine (S) on C-terminus of scSTING showed that both S388 and S396 were important for mediated signaling. Furthermore, scSTING overexpression inhibited ISKNV infection, and knockdown of scSTING promoted ISKNV infection, indicating that scSTING could suppress ISKNV infection in MFF-1 cells. These observations suggested that the scSTING played an important role in innate immune against ISKNV infection. Our work would help elucidate the roles of teleost fish STING in innate immunity.

Molecular cloning of Y-Box binding protein-1 from mandarin fish and its roles in stress-response and antiviral immunity

Mandarin fish (Siniperca chuatsi) is a universally farmed fish species in China and has a large farming scale and economic value. With the high-density cultural mode in mandarin fish, viral diseases, such as infectious spleen and kidney necrosis virus (ISKNV) and Siniperca chuatsi rhabdovirus (SCRV), have increased loss, which has seriously restricted the development of aquaculture. Y-Box binding protein 1 (YB-1) is a member of cold shock protein family that regulates multiple cellular processes. The roles of mammalian YB-1 protein in environmental stress and innate immunity have been studied well, but its roles in teleost fishes remain unknown. In the present study, the characteristic of S. chuatsi YB-1 (scYB-1) and its roles in cold stress and virus infection were investigated. The scYB-1 obtained an 1541 bp cDNA that contains a 903 bp open reading frame encoding a protein of 300 amino acids. Tissue distribution results showed that the scYB-1 is a ubiquitously expressed gene found among tissues from mandarin fish. Overexpression of scYB-1 can increase the expression levels of cold shock-responsive genes, such as scHsc70a, scHsc70b, and scp53. Furthermore, the role of scYB-1 in innate immunity was also investigated in mandarin fish fry (MFF-1) cells. The expression level of scYB-1 was significant change in response to poly (I:C), poly (dG:dC), PMA, ISKNV, or SCRV stimulation. The overexpression of scYB-1 can significantly increase the expression levels of NF-κB-responsive genes, including scIL-8, scTNF-α, and scIFN-h. The NF-κB-luciferase report assay results showed that the relative expression of luciferin was significantly increased in the cells overexpressed with scYB-1 compared with those in cells overexpressed with control plasmid. These results indicate that scYB-1 can induce the NF-κB signaling pathway in MFF-1 cells. Overexpressed scYB-1 can downregulate the expression of ISKNV viral major capsid protein (mcp) gene but upregulates the expression of SCRV mcp gene. Moreover, knockdown of scYB-1 using siRNA can upregulate the expression of ISKNV mcp gene but downregulates the expression of SCRV mcp gene. These results indicate that scYB-1 suppresses ISKNV infection while enhancing SCRV infection. The above observations suggest that scYB-1 is involved in cold stress and virus infection. Our study will provide an insight into the roles of teleost fish YB-1 protein in stress response and innate immunity.

Differential autophagic effects triggered by five different vertebrate iridoviruses in a common, highly permissive mandarinfish fry (MFF-1) cell model.

Autophagy of five vertebrate iridoviruses, including one megalocytivirus (infectious spleen and kidney necrosis virus, ISKNV) and four ranaviruses (Chinese giant salamander iridovirus, CGSIV; Tiger frog virus, TFV; Grouper iridovirus, GIV; and Largemouth bass virus, LMBV) were investigated in a common, highly permissive mandarinfish fry (MFF-1) cell model. The results showed marked autophagosome formation in GIV- and LMBV-infected cells but not in ISKNV-, CGSIV- and TFV-infected MFF-1cells. Strong evidence for the autophagosomes was provided by transmission electron microscopy, the detection of mandarinfish microtubule-associated protein 1 light chain 3B (mLC3)-based fluorescent dot formation and mLC3-I/mLC3-II conversion was provided by Western blotting. Pharmacological tests indicated that autophagy plays an antiviral role during GIV or LMBV infection. Collectively, our data are the first to show that antiviral autophagic effects can be triggered by GIV and LMBV but not by ISKNV, TFV and CGSIV in a common susceptible cell model. These results suggest that differential host-virus interaction strategies may be utilized against different vertebrate iridoviruses; they also indicate the potential effectiveness of an antiviral treatment that modulates autophagy to control iridoviral infections, such as GIV and LMBV.

Field trial tests of FKC vaccines against RSIV genotype Megalocytivirus in cage-cultured mandarin fish (Siniperca chuatsi) in an inland reservoir

Megalocytiviruses are one of the most important causative agents in finfish industry in China, Japan and South East Asia. The viruses are mainly composed of ISKNV, RSIV and TRBIV genotypes. Among them, ISKNV genotype isolate is the most important causative agent in mandarin fish industry in South China. Since its first occurrence in mid-1990s in China, no effective drug has been developed to prevent and control this virus until our recent work. In this study, unusual RSIV genotype Megalocytivirus was validated as the causative agent in natural mass mortality of cage-cultured mandarin fish in an inland reservoir. One isolate was obtained using MFF-1 cells from natural mass mortality of mandarin fish and designated as Megalocyti-LJ2012. Based on two previous megalocytiviral isolates, formalin-killed cell (FKC) vaccines were prepared to immunize 2000 and 9000 cage-cultured mandarin in October 2011 and August 2012, respectively. As results, greater than 70% protective effects were observed in vaccination group in both individual field tests. Adjuvant-emulsified FKC vaccine provided even greater than 99% protective effect (N = 1000). In contrast, almost all fish died in non-vaccination group (N = 1000). Immuno-protection test under laboratory condition showed that 100% relative percent survival was obtained in surviving fish from vaccination group after challenge with Megalocyti-LJ2012 at 4 months post vaccination. Taken together, the present study shows that FKC vaccine is also efficient in preventing RSIV genotype Megalocytivirus in cage-cultured mandarin fish in two field tests.

The potential role of microfilaments in host cells for infection with infectious spleen and kidney necrosis virus infection

BackgroundInfectious spleen and kidney necrosis virus (ISKNV) belongs to the genus Megalocytivirus from the family Iridoviridae. Megalocytivirus causes severe economic losses to tropical freshwater and marine culture industry in Asian countries and is devastating to the mandarin fish farm industry in China particularly.MethodsWe investigated the involvement of microfilaments in the early and late stages of ISKNV infection in MFF-1 cells by selectively perturbing their architecture using well-characterized inhibitors of actin dynamics. The effect of disruption of actin cytoskeleton on ISKNV infection was evaluated by indirect immunofluorescence analysis or real-time quantitative PCR.ResultsThe depolymerization of the actin filaments with cytochalasin D, cytochalasin B, or latrunculin A reduced ISKNV infection. Furthermore, depolymerization of filamentous actin by inhibitors did not inhibit binding of the virus but affected virus internalization in the early stages of infection. In addition, the depolymerization of actin filaments reduced total ISKNV production in the late stages of ISKNV.ConclusionsThis study demonstrated that ISKNV required an intact actin network during infection. The findings will help us to better understand how iridoviruses exploit the cytoskeleton to facilitate their infection and subsequent disease.

Co-infection of megalocytivirus and viral nervous necrosis virus in a very severe mass mortality of juvenile orange-spotted groupers (Epinephelus coioides)

Megalocytivirus is a large double-stranded DNA virus, and viral nervous necrosis virus (VNNV) is a piscine nodavirus comprising two single-stranded RNAs. Both of them are the major viral causative agents of grouper diseases worldwide. However, the co-infection of these two agents in the same mass mortality has not yet been reported. In mid-June 2011, a mass mortality of juvenile orange-spotted groupers occurred in a juvenile grouper rearing farm in Qionghai county, Hainan province, China. Approximately 270 000 juvenile groupers died in two weeks. The moribund fishes were sampled for viral agent detection by molecular investigation. Both megalocytivirus and VNNV were detected and designated as OGSIV-QH11 and GNNV-QH11, respectively. MCP/CP sequence analyses revealed that OGSIV-QH11 was an RSIV type of megalocytivirus and GNNV-QH11 was an RGNNV-type VNNV. Virus isolation was performed in the MFF-1 cell line, which is highly susceptible for megalocytivirus. OSGIV-QH11 can grow well in MFF-1 cells, as confirmed by transmission electron microscopy (TEM), indirect immunofluorescence assay and virus purification. GNNV-QH11 was detected in infected MFF-1 cells by RT-PCR but not by TEM. In summary, we first confirmed the co-infection of megalocytivirus and viral nervous necrosis virus in a mass mortality of juvenile orange-spotted groupers, and then isolated a megalocytivirus from the infected MFF-1 cells. Our study provides a better understanding of viral diseases in grouper aquaculture industry.

Infectious Spleen and Kidney Necrosis Virus (a Fish Iridovirus) Enters Mandarin Fish Fry Cells via Caveola-Dependent Endocytosis

Infectious spleen and kidney necrosis virus (ISKNV) is the type species of the genus Megalocytivirus from the family Iridoviridae. Megalocytiviruses have been implicated in more than 50 fish species infections and currently threaten the aquaculture industry, causing great economic losses in China, Japan, and Southeast Asia. However, the cellular entry mechanisms of megalocytiviruses remain largely uncharacterized. In this study, the main internalization mechanism of ISKNV was investigated by using mandarin fish fry (MFF-1) cells. The progression of ISKNV infection is slow, and infection is not inhibited when the cells are treated with ammonium chloride (NH(4)Cl), chloroquine, sucrose, and chlorpromazine, which are inhibitors of clathrin-dependent endocytosis. The depletion of cellular cholesterol by methyl-β-cyclodextrin results in the significant inhibition of ISKNV infection; however, the infection is resumed with cholesterol replenishment. Inhibitors of caveolin-1-involved signaling events, including phorbol 12-myristate 13-acetate (PMA), genistein, and wortmannin, impair ISKNV entry into MFF-1 cells. Moreover, ISKNV entry is dependent on dynamin and the microtubule cytoskeleton. Cofraction analysis of ISKNV and caveolin-1 showed that ISKNV colocates with caveolin-1 during virus infection. These results indicate that ISKNV entry into MFF-1 cells proceeds via classical caveola-mediated endocytosis and is dependent on the microtubules that serve as tracks along which motile cavicles may move via a caveola-caveosome-endoplasmic reticulum (ER) pathway. As a fish iridovirus, ISKNV entry into MFF-1 cells is different from the clathrin-mediated endocytosis of frog virus 3 entry into mammalian cells (BHK-21) at 28°C, which has been recognized as a model for iridoviruses. Thus, our work may help further the understanding of the initial steps of iridovirus infection.

Antigenic identification of virion structural proteins from infectious spleen and kidney necrosis virus

Infectious spleen and kidney necrosis virus (ISKNV), belonging to the genus Megalocytivirus in the family Iridoviridae, is one of the major agents causing mortality and economic losses to the freshwater fish culture industry in Asian countries. Currently, little information regarding the antigenic properties of Megalocytivirus (especially ISKNV) is available. Our previous study using four different workflows with systematic and comprehensive proteomic approaches led to the identification of 38 ISKNV virion-associated proteins (J. Virol. 2869–2877, 2011). Thus, in this report, the antigenicity of 31 structural proteins from ISKNV virion was investigated. A one-dimensional gel electrophoresis immunoblot profile coupled with MALDI-TOF-TOF MS/MS was applied to identify six immunogenic viral proteins, namely, ORFs major capsid protein (006L), 054L, 055L, 101L, 117L, and 125L. Then, the antigenicity of 31 structural proteins was characterized by Western blot by using pooled sera from mandarin fish that survived ISKNV infection. Of the 31 viral proteins, 22 were recognized by the fish ISKNV antiserum. Furthermore, this antiserum neutralizes MFF-1 cells ISKNV infection. To our knowledge, this study is the first report on the immunogenicity of viral proteins and characterization of the proteome of megalocytivirus infective agents. Our findings are expected to promote the development of effective vaccine candidates.

Involvement of caveolin-1 in the Jak–Stat signaling pathway and infectious spleen and kidney necrosis virus infection in mandarin fish ( Siniperca chuatsi)

Caveolae, the major source of caveolin-1 protein, are specialized invaginated microdomains of the plasma membrane that act as organizing centers for signaling molecules in the immune system. In the present study, we report the cloning and characterization of caveolin-1 (mCav-1) from mandarin fish ( Siniperca chuatsi) and study on the roles of mCav-1 in the fish Jak–Stat signaling pathway and in virus infection. The cDNA sequence of mCav-1 was 707 bp in size, encoding a protein of 181 amino acids, which was different from the mammalian protein (178 amino acids). The deduced amino acid sequence of mCav-1 shared similar architecture with vertebrate caveolin-1 proteins, but mCav-1 lacked a phosphorylation site (y14). The major subcellular location of mCav-1 was in the caveolae, where the protein appeared to have major functions. Real-time PCR revealed that the expression of the mandarin fish Mx, IRF-1, SOCS1, and SOCS3 genes involved in the poly(I:C)-induced Jak–Stat signaling pathway was impaired by the mCav-1 scaffolding domain peptide (mSDP). In mandarin fish fry (MFF-1) cells, the protein levels of mCav-1 were markedly up-regulated at 12 and 24 h post-infection with ISKNV (infectious spleen and kidney necrosis virus). In addition, ISKNV entry into MFF-1 cells was significantly inhibited by mSDP, and the inhibition was dose-dependent. Thus, ISKNV infection was apparently associated with mCav-1 protein and may utilize the caveolae-related endocytosis pathway. The findings reported here further our understanding of the function of caveolin-1 in the complex signal transduction network in fish immune systems and in the cellular entry mechanism of iridoviruses.

A new marine megalocytivirus from spotted knifejaw, Oplegnathus punctatus, and its pathogenicity to freshwater mandarinfish, Siniperca chuatsi

Megalocytivirus is a newly defined piscine iridovirus and has been shown to be an important causative agent of viral diseases in fish. Here, a new megalocytivirus strain, designated SKIV-ZJ07, was isolated from spotted knifejaw ( Oplegnathus punctatus) using a mandarinfish fry cell line (MFF-1). Phylogenetic analysis of the major capsid protein and ATPase genes showed that SKIV-ZJ07 was most similar to the orange-spotted grouper iridovirus (OSGIV) from China and a U1 strain red sea bream iridovirus (RSIV-U1) from Japan. SKIV-ZJ07 was purified and the major viral proteins were identified using one-dimensional gel electrophoresis mass spectrometry (1-DE-MS) analysis. Twenty proteins were found to match proteins derived from rock sea bream iridovirus (RBIV), OSGIV and infectious spleen and kidney necrosis virus (ISKNV). Among these, 19 proteins had not been previously identified as virion-associated proteins in megalocytivirus. Challenge tests showed that SKIV-ZJ07 was highly virulent in mandarinfish. Infected fish displayed typical histopathological symptoms of ISKNV-infected fish and died, indicating that the mandarinfish is an ideal model for further study of megalocytivirus–host interactions, molecular mechanisms of viral infection and pathogenesis. Interestingly, large numbers of regular paracrystalline SKIV-ZJ07 virion arrays were observed in both SKIV-infected MFF-1 cells and mandarinfish tissues by transmission electron microscopy (TEM), which is unusual for megalocytivirus under artificial infection conditions. Taken together, the results presented here provide new insight into the pathology of megalocytivirus infection.

Development of a mandarin fish Siniperca chuatsi fry cell line suitable for the study of infectious spleen and kidney necrosis virus (ISKNV)

Infectious spleen and kidney necrosis virus (ISKNV) is a typical species of the genus Megalocytivirus in the family Iridoviridae. However, until recently, no suitable piscine cell line is stably susceptible to ISKNV. Here, a continuous cell culture derived from the mandarin fish fry (MFF-1) was developed and has been subcultured over 60 passages during a period of 18 months. MFF-1 consists predominantly of epithelial-like cells and grows well in DMEM supplemented with 10% fetal bovine serum. MFF-1 could produce high titers of ISKNV by continuous viral passages which were further confirmed by indirect immunofluorescence assay and RT-PCR analysis. Flow cytometry analyse showed that approximately 80.3% cells could be infected by ISKNV at 3 days post-infection. Abundant ISKNV particles were observed in the cytoplasm of the ISKNV infected MFF-1 cells by transmission electron microscopy. Mandarin fish injected with the filtrate from the infected cell suspension developed clinical signs and died, which is in accordance with the infectious spleen and kidney necrosis virus disease (ISKNVD). In addition, apoptosis was observed in the MFF-1 cells upon ISKNV infection by FITC-annexin V staining. ISKNV was purified and the viral protein profiles were also determined in this research. To our knowledge, MFF-1 is the first cell line originated from mandarin fish and it can be an efficient tool for the study of ISKNV.