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Li Sun - One of the best experts on this subject based on the ideXlab platform.

  • Phosphatase and Tensin Homolog (PTEN) of Japanese Flounder-Its Regulation by miRNA and Role in Autophagy, Apoptosis and Pathogen Infection.
    International journal of molecular sciences, 2020
    Co-Authors: Xiao-lu Guan, Li Sun
    Abstract:

    MicroRNAs (miRNAs) are small non-coding RNAs with important roles in diverse biological processes including immunity. Japanese flounder (Paralichthys olivaceus) is an aquaculture fish species susceptible to the infection of bacterial and viral pathogens including Edwardsiella tarda. In a previous study, pol-miR-novel_547, a novel miRNA of flounder with unknown function, was found to be induced by E. tarda. In the present study, we investigated the regulation and function of pol-miR-novel_547 and its target gene. We found that pol-miR-novel_547 was regulated differently by E. tarda and the viral pathogen Megalocytivirus, and pol-miR-novel_547 repressed the expression of PTEN (phosphatase and tensin homolog) of flounder (PoPTEN). PoPTEN is ubiquitously expressed in multiple tissues of flounder and responded to bacterial and viral infections. Interference with PoPTEN expression in flounder cells directly or via pol-miR-novel_547 promoted E. tarda invasion. Consistently, in vivo knockdown of PoPTEN enhanced E. tarda dissemination in flounder tissues, whereas in vivo overexpression of PoPTEN attenuated E. tarda dissemination but facilitated Megalocytivirus replication. Further in vitro and in vivo studies showed that PoPTEN affected autophagy activation via the AKT/mTOR pathway and also modulated the process of apoptosis. Together these results reveal for the first time a critical role of fish PTEN and its regulatory miRNA in pathogen infection, autophagy, and apoptosis.

  • Transcriptome analysis reveals seven key immune pathways of Japanese flounder (Paralichthys olivaceus) involved in Megalocytivirus infection.
    Fish & shellfish immunology, 2020
    Co-Authors: Xianhui Ning, Shuai Jiang, Li Sun
    Abstract:

    Megalocytivirus is a serious viral pathogen to many farmed fish including Japanese flounder (Paralichthys olivaceus). In this study, in order to systematically identify host immune genes induced by Megalocytivirus infection, we examined the transcription profiles of flounder infected by Megalocytivirus for 2, 6, and 8 days. Compared with uninfected fish, virus-infected fish exhibited 1242 differentially expressed genes (DEGs), with 225, 275, and 877 DEGs occurring at 2, 6, and 8 days post infection, respectively. Of these DEGs, 728 were upregulated and 659 were downregulated. The majority of DEGs were time-specific and formed four distinct expression profiles well correlated with the time of infection. The DEGs were classified into diverse Gene Ontology (GO) functional terms and enriched in 27 Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways, approximately one third of which were related to immunity. Weighted co-expression network analysis (WGCNA) was used to identify 16 key immune DEGs belonging to seven immune pathways (RIG-I-like receptor signaling pathway, JAK-STAT signaling pathway, TLR signaling pathway, cytokine-cytokine receptor interaction, phagosome, apoptosis, and p53 signaling pathway). These pathways interacted extensively and formed complicated networks. This study provided a global picture of Megalocytivirus-induced gene expression profiles of flounder at the transcriptome level and uncovered a set of key immune genes and pathways closely linked to Megalocytivirus infection. These results provided a set of targets for future delineation of the key factors implicated in the anti-Megalocytivirus immunity of flounder.

  • Global profiling of Megalocytivirus-induced proteins in tongue sole (Cynoglossus semilaevis) spleen identifies cellular processes essential to viral infection
    Developmental and comparative immunology, 2018
    Co-Authors: Jian Zhang, Li Sun
    Abstract:

    Megalocytivirus is a DNA virus with a broad host range among farmed fish including tongue sole (Cynoglossus semilaevis). In this study, label-free proteomics was performed to examine protein expression in tongue sole spleen induced by Megalocytivirus at 8 and 12 days post infection (dpi). Compared to uninfected control fish, virus-infected fish displayed 315 up-regulated proteins and 111 down-regulated proteins at 8 dpi, and 48 up-regulated proteins and 43 down-regulated proteins at 12 dpi. The expressions of five differentially expressed proteins were confirmed by Western blot. The differentially expressed proteins were enriched in the pathways and processes associated with innate immune response and viral infection. Interference with the expression of two up-regulated proteins of the ubiquitin proteasome system (UPS), i.e. proteasome assembly chaperone 2 and proteasome maturation protein, significantly reduced viral propagation in fish, whereas overexpression of these two proteins significantly enhanced viral propagation. Consistently, inhibition of the functioning of proteasome significantly impaired viral replication in vivo. This study provided the first global protein profile responsive to Megalocytivirus in tongue sole, and revealed an essential role of UPS in viral infection.

  • Cynoglossus semilaevis ISG15: a secreted cytokine-like protein that stimulates antiviral immune response in a LRGG motif-dependent manner. PLoS One 2012; 7: e44884
    2016
    Co-Authors: Wei Wang, Min Zhang, Zhizhong Xiao, Li Sun
    Abstract:

    ISG15 is an ubiquitin-like protein that is induced rapidly by interferon stimulation. Like ubiquitin, ISG15 forms covalent conjugates with its target proteins in a process called ISGylation, which in mammals is known to play a role in antiviral immunity. In contrast to mammalian ISG15, the function of teleost ISG15 is unclear. In this study, we identified and analyzed the function of an ISG15 homologue, CsISG15, from tongue sole (Cynoglossus semilaevis). CsISG15 is composed of 162 residues and possesses two tandem ubiquitin-like domains and the highly conserved LRGG motif found in all known ISG15. Expression of CsISG15 occurred in a wide range of tissues and was upregulated in kidney and spleen by viral and bacterial infection. In vitro study with primary head kidney (HK) lymphocytes showed that Megalocytivirus infection caused induction of CsISG15 expression and extracellular release of CsISG15 protein. Purified recombinant CsISG15 (rCsISG15) activated HK macrophages and enhanced the expression of immune genes in HK lymphocytes, both these effects, however, were significantly reduced when the conserved LRGG sequence was mutated to LAAG. Further study showed that the presence of rCsISG15 during Megalocytivirus infection of HK lymphocytes reduced intracellular viral load, whereas antibody blocking of CsISG15 enhanced viral infection. Likewise, interference with CsISG15 expression by RNAi promoted viral infection. Taken together, these results indicate that CsISG15, a teleost ISG15, promotes antiviral immune response and that, unlike mammalian ISG15, CsISG15 exerts its immunoregulatory effect in the form of an unconjugated extracellular cytokine. I

  • pol-miR-731, a teleost miRNA upregulated by Megalocytivirus, negatively regulates virus-induced type I interferon response, apoptosis, and cell cycle arrest
    Scientific reports, 2016
    Co-Authors: Bao-cun Zhang, Ze-jun Zhou, Li Sun
    Abstract:

    Megalocytivirus is a DNA virus that is highly infectious in a wide variety of marine and freshwater fish, including Japanese flounder (Paralichthys olivaceus), a flatfish that is farmed worldwide. However, the infection mechanism of Megalocytivirus remains largely unknown. In this study, we investigated the function of a flounder microRNA, pol-miR-731, in virus-host interaction. We found that pol-miR-731 was induced in expression by Megalocytivirus and promoted viral replication at the early infection stage. In vivo and in vitro studies revealed that pol-miR-731 (i) specifically suppresses the expression of interferon regulatory factor 7 (IRF7) and cellular tumor antigen p53 in a manner that depended on the integrity of the pol-miR-731 complementary sequences in the 3' untranslated regions of IRF7 and p53, (ii) disrupts Megalocytivirus-induced Type I interferon response through IRF7, (iii) inhibits Megalocytivirus-induced splenocyte apoptosis and cell cycle arrest through p53. Furthermore, overexpression of IRF7 and p53 abolished both the inhibitory effects of pol-miR-731 on these biological processes and its stimulatory effect on viral replication. These results disclosed a novel evasion mechanism of Megalocytivirus mediated by a host miRNA. This study also provides the first evidence that a virus-induced host miRNA can facilitate viral infection by simultaneously suppressing several antiviral pathways.

Shaoping Weng - One of the best experts on this subject based on the ideXlab platform.

  • Identification of infectious spleen and kidney necrosis virus (ISKNV)-encoded microRNAs.
    Virus genes, 2020
    Co-Authors: Qiong Xia, Shaoping Weng
    Abstract:

    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.

  • Interaction of Infectious Spleen and Kidney Necrosis Virus ORF119L with PINCH Leads to Dominant-Negative Inhibition of Integrin-Linked Kinase and Cardiovascular Defects in Zebrafish
    Journal of virology, 2014
    Co-Authors: Ji-min Yuan, Chang-jun Guo, Lu-yun Yang, Shaoping Weng
    Abstract:

    Infectious spleen and kidney necrosis virus (ISKNV) is the type species of the Megalocytivirus genus, Iridoviridae family, causing a severe systemic disease with high mortality in mandarin fish (Siniperca chuatsi) in China and Southeast Asia. At present, the pathogenesis of ISKNV infection is still not fully understood. Based on a genome-wide bioinformatics analysis of ISKNV-encoded proteins, we found that ISKNV open reading frame 119L (ORF119L) is predicted to encode a three-ankyrin-repeat (3ANK)-domain-containing protein, which shows high similarity to the dominant negative form of integrin-linked kinase (ILK); i.e., viral ORF119L lacks the ILK kinase domain. Thus, we speculated that viral ORF119L might affect the host ILK complex. Here, we demonstrated that viral ORF119L directly interacts with particularly interesting Cys-His-rich protein (PINCH) and affects the host ILK-PINCH interaction in vitro in fathead minnow (FHM) cells. In vivo ORF119L overexpression in zebrafish (Danio rerio) embryos resulted in myocardial dysfunctions with disintegration of the sarcomeric Z disk. Importantly, ORF119L overexpression in zebrafish highly resembles the phenotype of endogenous ILK inhibition, either by overexpressing a dominant negative form of ILK or by injecting an ILK antisense morpholino oligonucleotide. Intriguingly, ISKNV-infected mandarin fish develop disorganized sarcomeric Z disks in cardiomyocytes. Furthermore, phosphorylation of AKT, a downstream effector of ILK, was remarkably decreased in ORF119L-overexpressing zebrafish embryos. With these results, we show that ISKNV ORF119L acts as a domain-negative inhibitor of the host ILK, providing a novel mechanism for the Megalocytivirus pathogenesis. IMPORTANCE Our work is the first to show the role of a dominant negative inhibitor of the host ILK from ISKNV (an iridovirus). Mechanistically, the viral ORF119L directly binds to the host PINCH, attenuates the host PINCH-ILK interaction, and thus impairs ILK signaling. Intriguingly, ORF119L-overexpressing zebrafish embryos and ISKNV-infected mandarin fish develop similar disordered sarcomeric Z disks in cardiomyocytes. These findings provide a novel mechanism for Megalocytivirus pathogenesis.

  • ORF005L from infectious spleen and kidney necrosis virus is located in the inner mitochondrial membrane and induces apoptosis.
    Virus genes, 2014
    Co-Authors: Rui Wang, Lihong Liu, Shaoping Weng
    Abstract:

    Infectious spleen and kidney necrosis virus (ISKNV) belongs to the genus Megalocytivirus in the family Iridoviridae. This virus is the etiological agent of a serious and pandemic disease in fish. Cells infected with ISKNV undergo apoptosis. In this study, the ISKNV ORF005L gene was characterized and functionally investigated. Bioinformatics analysis revealed that the ORF005L protein contains a region similar to the catalytic domain of CTD-like phosphatases. Real-time quantitative-PCR results showed the transcription profile of ORF005L in ISKNV-infected cells; in these cells, ORF005L was initially transcribed at 24 h post-infection. The green fluorescent protein-tagged ORF005L protein was localized in the mitochondria. Sub-mitochondrial fractions were subjected to Western blot, and the results showed that ORF005L was specifically located in the inner membrane of the mitochondria. The ORF005L in fathead minnow cells was transiently expressed, resulting in the decrease in mitochondrial transmembrane potential, which induced cell apoptosis. ORF005L was knocked down by specific dsRNA, thereby significantly reducing the apoptosis of mandarin fish fry-1 cells induced by ISKNV infection. These results indicated that the ORF005L of ISKNV could disrupt mitochondrial function and cause apoptosis. This study may provide novel insights into the pathogenesis of Megalocytivirus infection.

  • Cloning of a new fibroblast cell line from an early primary culture from mandarin fish (Siniperca chuatsi) fry for efficient proliferation of Megalocytiviruses.
    Cytotechnology, 2013
    Co-Authors: Chuanfu Dong, Fan Shuang, Shaoping Weng
    Abstract:

    Megalocytiviruses are important emerging pathogens in both freshwater and marine finfish aquaculture. However, a limited number of piscine cell lines are persistently susceptible to these viruses, which greatly limits the study of Megalocytiviruses. In this study, a new fibroblast-like cell line was established from an early primary culture from mandarin fish fry by a single cell cloning and was designated as MFF-8C1. The MFF-8C1 cells grow well in Dulbecco’s modified Eagle’s medium supplemented with 10 % fetal bovine serum and had been subcultured more than 60 passages since the initial recovery culture in October 2009. Chromosomal analysis revealed that 91 % of the MFF-8C1 cells maintained a normal diploid chromosome number (2n = 48) in the 46th passage. Infection experiments showed that both freshwater-borne and marine-borne Megalocytiviruses induce severe cytopathic effects in infected MFF-8C1 cells characterized by the rounding and enlargement of cells, which are highly consistent with the previous description of the infection in other susceptible cells with Megalocytivirus. Megalocytivirus infections were further confirmed by a transmission electron microscopy. Furthermore, the MFF-8C1-cultured megalocytiviral suspension was highly virulent to infected mandarin fish. In summary, a new fibroblast cell line from mandarin fish fry that was highly permissive to Megalocytiviruses was established. The MFF-8C1 cell line is a promising cellular substrate candidate for cell-cultured vaccine production of Megalocytivirus.

  • Field trial tests of FKC vaccines against RSIV genotype Megalocytivirus in cage-cultured mandarin fish (Siniperca chuatsi) in an inland reservoir.
    Fish & shellfish immunology, 2013
    Co-Authors: Youyong Dong, Shaoping Weng, Chuanfu Dong
    Abstract:

    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.

Hyun Do Jeong - One of the best experts on this subject based on the ideXlab platform.

  • Development of a high-dose vaccine formulation for prevention of Megalocytivirus infection in rock bream (Oplegnathus fasciatus).
    Vaccine, 2020
    Co-Authors: Woo Ju Kwon, Suhee Hong, Young Chul Kim, Kwang Il Kim, Joon Bum Jeong, Jae Chan Choi, Min Gyeong Jeong, Joon Gyu Min, Hyun Do Jeong
    Abstract:

    Abstract A formalin-inactivated red sea bream iridovirus (RSIV) vaccine was prepared using the culture supernatant of a persistently infected Pagrus major fin cell line (PI-PMF) with IVS-1 strain (RSIV subtype II Meglaocytivirus). Rock bream (Oplegnathus fasciatus) were injected with a high-dose, ultracentrifuged Megalocytivirus vaccine (Ultra HSCMV, 7.0 × 1010 copies/mL), a high-dose supernatant of cultured Megalocytivirus vaccine (HSCMV, 1.0 × 1010 copies/mL), a supernatant of cultured Megalocytivirus vaccine (SCMV, 1.0 × 109 copies/mL), and a low-dose of cultured Megalocytivirus vaccine (LSCMV, 1.0 × 108 copies/mL). The vaccine efficacies for the various vaccine formulations were determined done following injection challenge with IVS-1 (1.0 × 104 copies/0.1 mL/fish), and the four different vaccines exhibited cumulative mortalities of 10.0 ± 0.0%, 48.3 ± 7.6%, 75.0 ± 5.0%, and 100.0 ± 0.0%, respectively. Additionally, the dose-dependent vaccine efficacy was also confirmed using two different cohabitation methods that included challenges G (general) and I (individual). When squalene + aluminum hydroxide (SqAl) was used as an adjuvant for the HSCMV or SCMV vaccine, cumulative mortalities of 30.0 ± 5.0% and 48.3 ± 7.6%, respectively, were obtained; moreover, these two adjuvants exhibited the highest efficacy in this study. The observed difference in survival post-challenge for the different vaccine concentrations was not reflected in the differences in neutralizing antibody titers. It was found that the water temperature during immune induction plays a less important a role than the water temperature during the challenge test, in which lowering the water temperature from 25 °C to 21 °C during a challenge improved the level of protection from cumulative mortalities from 35% to 10%. This study demonstrated that protection against mortality using inactivated vaccines against RSIVD in rock bream, which are known to be the most susceptible species to RSIV infection, is dependent upon antigen dose and temperature during the challenge.

  • Development and characterization of Megalocytivirus persistently-infected cell cultures for high yield of virus.
    Tissue & cell, 2020
    Co-Authors: Woo Ju Kwon, Ji Woong Jin, Suhee Hong, Young Chul Kim, Min Ji Yoon, Kwang Il Kim, Joon Bum Jeong, Hyun Do Jeong
    Abstract:

    Abstract Megalocytivirus infection is a major threat in rock bream aquaculture in Korea. To produce a highly concentrated Megalocytivirus, primary cells, established cell line and persistently infected cell line were used in this study. Megalocytivirus was inoculated in primary fin cell cultures of red sea bream (Pagrus major), rock bream (Oplegnathus fasciatus), olive flounder (Paralichthys olivaceus) and black sea bream (Acanthopagrus schlegelii) and produced at similar concentrations of 108.99 − 9.88 viral particles/mL in all cultures while produced 107.31 viral particles/mL in grunt fin (GF) cell line. Since only red sea bream fin culture was amenable to subculturing for more than 100 times, it was established into Pagrus major fin (PMF) cell line. A persistently infected PMF cell line (PI-PMF) was obtained by continuous subculturing every 7 days as a batch culture system (PI-PMF-B) after infecting with Megalocytivirus. Virus in supernatant of PI-PMF-B was maintained at high concentrations throughout over 50 consecutive subcultures in a relatively narrow range from 108.33 to 108.94 viral particles/mL with high level of CPE. For a more efficient and convenient production, a semi-batch culture system (PI-PMF-S) was developed in which culture media were exchanged at intervals of 3 days without subculturing for more than 50 media exchanges. Despite low virus productivity in a single cell (specific virus productivity, SVP), total cell number was increased in PI-PMF-S, allowing us to efficiently obtain a much higher concentration of virus (108.56 to 109.75 viral particles/mL) than in PMF-B. This is the first study to report detailed new methods for continuous and efficient production of high concentrations of megalocytivivrus with characterization of viral propagation in persistently infected cells.

  • Genetic diversity of Megalocytivirus from cultured fish in Korea
    Aquaculture, 2019
    Co-Authors: Kwang Il Kim, Woo Ju Kwon, Eun Sun Lee, Mi-young Cho, Sung Hee Jung, Seong Don Hwang, Jeong Wan, Bo Young Jee, Hyun Do Jeong
    Abstract:

    Abstract Since the first outbreak of Megalocytivirus infection in cultured marine fish in the 1990s, the Red sea bream iridovirus (RSIV) subtype II and Turbot reddish body iridovirus (TRBIV) type Megalocytivirus have been prevalent in Korea. Based on the major capsid protein (MCP) genes, the RSIV subtype II, as the predominant genotype, was identified in marine and freshwater fish including thread-sail filefish (Stephanolepis cirrhifer), so-iuy mullet (Planiliza haematocheila), starry flounder (Platichthys stellatus), and golden mandarin fish (Siniperca scherzeri). Recently, a new genetic variant of RSIV type (subtype I and II), SB5-TY originating from sea bass (Lateolabrax japonicas), was identified by their sequence identity with the four ankyrin repeat domains of RSIV (401R, 424R, 534L, and 641L). This result corroborate the evidence that increased genotypic variation in RSIV-type Megalocytiviruses has been detected.

  • A natural infection by the red sea bream iridovirus-type Megalocytivirus in the golden mandarin fish Siniperca scherzeri.
    Journal of fish diseases, 2018
    Co-Authors: Kwang Il Kim, Young Chul Kim, Mi-young Cho, Sung Hee Jung, Seong Don Hwang, Hyun Do Jeong
    Abstract:

    An outbreak of a Megalocytivirus infection was found in the golden mandarin fish Siniperca scherzeri during September and October 2016, in Korea. Phylogeny and genetic diversity based on the major capsid protein (MCP) and adenosine triphosphatase (ATPase) genes showed a new strain. Designated as GMIV, this strain derived from the golden mandarin fish was suggested to belong to the red sea bream iridovirus (RSIV)-subgroup I. Additionally, this train clustered with the ehime-1 strain from red sea bream Pagrus major in Japan and was distinguished from circulating isolates (RSIV-type subgroup II and turbot reddish body iridovirus [TRBIV] type) in Korea. The infection level, evaluated by qPCR, ranged from 8.18 × 102 to 7.95 × 106  copies/mg of tissue individually, suggesting that the infected fish were in the disease-transmitting stage. The diseased fish showed degenerative changes associated with cytomegaly in the spleen as general sign of Megalocytivirus infection. The results confirm that the RSIV-type Megalocytivirus might have crossed the environmental and species barriers to cause widespread infection in freshwater fish.

  • Identification and Characterization of Megalocytivirus Type 3 Infection with Low Mortality in Starry Flounder, Platichthys stellatus, in Korea
    Journal of the World Aquaculture Society, 2017
    Co-Authors: Ji Woong Jin, Yi Kyung Kim, Suhee Hong, Young Chul Kim, Woo Ju Kwon, Hyun Do Jeong
    Abstract:

    MVSF-12 belonging to Megalocytivirus type 3 was isolated from cultured starry flounder; Platichthys stellatus, at the moribund or subclinical stage with low mortalities in Korea. Of 20 apparently healthy fish in the farms, 17 were also confirmed in nested polymerase chain reaction to be infected by Megalocytivirus. When starry flounder; rock bream, Oplegnathus fasciatus; and olive flounder, Paralichthys olivaceus, were artificially infected by MVSF-12 or iridovirus sachun-1 (IVS-1, Megalocytivirus type 1), starry flounder and olive flounder showed no mortality until Day 24, without any clinical signs including enlarged spleen, while rock bream showed 100% mortality by IVS-1 infection within 11 d but no mortality by MVSF-12. Although it was not pathogenic, MVSF-12 in infected fish at Day 24 was viable when successfully cultured in vitro using primary rock bream embryo cells and produced a cytopathic effect (CPE) with the viral copy numbers between 1.76 × 107 and 5.23 × 107/mL of culture supernatant. In conclusion, this study demonstrates the low pathogenicity of MVSF-12 and low susceptibility of starry flounder and olive flounder to both MVSF-12 and IVS-1. Indeed, MVSF-12 at the subclinical stage could be replicated with CPE in vitro, indicating a possibility to induce pathogenic effects and mortality under adverse environment or physiologic conditions.

Chuanfu Dong - One of the best experts on this subject based on the ideXlab platform.

  • Cloning of a new fibroblast cell line from an early primary culture from mandarin fish (Siniperca chuatsi) fry for efficient proliferation of Megalocytiviruses.
    Cytotechnology, 2013
    Co-Authors: Chuanfu Dong, Fan Shuang, Shaoping Weng
    Abstract:

    Megalocytiviruses are important emerging pathogens in both freshwater and marine finfish aquaculture. However, a limited number of piscine cell lines are persistently susceptible to these viruses, which greatly limits the study of Megalocytiviruses. In this study, a new fibroblast-like cell line was established from an early primary culture from mandarin fish fry by a single cell cloning and was designated as MFF-8C1. The MFF-8C1 cells grow well in Dulbecco’s modified Eagle’s medium supplemented with 10 % fetal bovine serum and had been subcultured more than 60 passages since the initial recovery culture in October 2009. Chromosomal analysis revealed that 91 % of the MFF-8C1 cells maintained a normal diploid chromosome number (2n = 48) in the 46th passage. Infection experiments showed that both freshwater-borne and marine-borne Megalocytiviruses induce severe cytopathic effects in infected MFF-8C1 cells characterized by the rounding and enlargement of cells, which are highly consistent with the previous description of the infection in other susceptible cells with Megalocytivirus. Megalocytivirus infections were further confirmed by a transmission electron microscopy. Furthermore, the MFF-8C1-cultured megalocytiviral suspension was highly virulent to infected mandarin fish. In summary, a new fibroblast cell line from mandarin fish fry that was highly permissive to Megalocytiviruses was established. The MFF-8C1 cell line is a promising cellular substrate candidate for cell-cultured vaccine production of Megalocytivirus.

  • Field trial tests of FKC vaccines against RSIV genotype Megalocytivirus in cage-cultured mandarin fish (Siniperca chuatsi) in an inland reservoir.
    Fish & shellfish immunology, 2013
    Co-Authors: Youyong Dong, Shaoping Weng, Chuanfu Dong
    Abstract:

    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.

  • Virions proteins of an RSIV-type Megalocytivirus from spotted knifejaw Oplegnathus punctatus (SKIV-ZJ07)
    Virology, 2013
    Co-Authors: Fan Shuang, Shaoping Weng, Yongwen Luo, Xiao-peng Xiong, Chuanfu Dong
    Abstract:

    Megalocytiviruses have three main genotypes, which are represented by ISKNV, RSIV, and TRBIV. To date, the virion-associated proteins of RSIV and TRBIV are still unknown. The spotted knifejaw iridovirus (SKIV) is a newly characterized RSIV-type Megalocytivirus. In this study, the virion-associated proteins of SKIV were identified by systemic one-dimensional gel electrophoresis-based proteomic approaches. A total of 49 viral proteins and 33 cellular proteins were associated with the SKIV virions by LC MS/MS, including 18 highly abundant structural proteins that were detected by MALDI TOF/TOF-MS. One highly abundant structural protein of interest was identified as the virus-inducible stress protein (VISP) and further characterized as an envelope protein. However, knockdown of mVISP by siRNA method showed no effect in virion production. The current study is the first to present detailed information on the virion-associated proteins of an RSIV-type Megalocytivirus and to identify a novel cellular envelope protein of this virus.

  • Antigenic identification of virion structural proteins from infectious spleen and kidney necrosis virus
    Fish & shellfish immunology, 2011
    Co-Authors: Xiao-peng Xiong, Shaoping Weng, Chuanfu Dong, Jing Zhang, Ye Zhang
    Abstract:

    Abstract 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.

  • A new marine Megalocytivirus from spotted knifejaw, Oplegnathus punctatus, and its pathogenicity to freshwater mandarinfish, Siniperca chuatsi.
    Virus research, 2009
    Co-Authors: Chuanfu Dong, Shaoping Weng, Yongwen Luo, Mingming Huang, Zhi-xin Yin
    Abstract:

    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.

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  • Global profiling of Megalocytivirus-induced proteins in tongue sole (Cynoglossus semilaevis) spleen identifies cellular processes essential to viral infection
    Developmental and comparative immunology, 2018
    Co-Authors: Jian Zhang, Li Sun
    Abstract:

    Megalocytivirus is a DNA virus with a broad host range among farmed fish including tongue sole (Cynoglossus semilaevis). In this study, label-free proteomics was performed to examine protein expression in tongue sole spleen induced by Megalocytivirus at 8 and 12 days post infection (dpi). Compared to uninfected control fish, virus-infected fish displayed 315 up-regulated proteins and 111 down-regulated proteins at 8 dpi, and 48 up-regulated proteins and 43 down-regulated proteins at 12 dpi. The expressions of five differentially expressed proteins were confirmed by Western blot. The differentially expressed proteins were enriched in the pathways and processes associated with innate immune response and viral infection. Interference with the expression of two up-regulated proteins of the ubiquitin proteasome system (UPS), i.e. proteasome assembly chaperone 2 and proteasome maturation protein, significantly reduced viral propagation in fish, whereas overexpression of these two proteins significantly enhanced viral propagation. Consistently, inhibition of the functioning of proteasome significantly impaired viral replication in vivo. This study provided the first global protein profile responsive to Megalocytivirus in tongue sole, and revealed an essential role of UPS in viral infection.

  • ORF75 of Megalocytivirus RBIV-C1: A global transcription regulator and an effective vaccine candidate.
    Fish & shellfish immunology, 2015
    Co-Authors: Jian Zhang
    Abstract:

    Megalocytivirus, a DNA virus belonging to the Iridoviridae family, is a severe pathogen to a wide range of marine and freshwater fish. In this study, using turbot (Scophthalmus maximus) as a host model, we examined the immunoprotective property of one Megalocytivirus gene, ORF75, in the form of DNA vaccine (named pORF75). Immunofluorescence microscopy and RT-PCR analysis showed that P444, the protein encoded by ORF75, was naturally produced in the tissues of turbot during Megalocytivirus infection, and that the vaccine gene in pORF75 was expressed in fish cells transfected with pORF75 and in the tissues of turbot immunized with pORF75. Following vaccination of turbot with pORF75, a high level of survival (73%) was observed against a lethal Megalocytivirus challenge. Consistently, viral replication in the vaccinated fish was significantly inhibited. Immune response analysis showed that pORF75-vaccinated fish (i) exhibited upregulated expression of the genes involved in innate and adaptive immunity, (ii) possessed specific memory immune cells that showed significant response to secondary antigen stimulation, and (iii) produced specific serum antibodies which, when co-introduced into turbot with Megalocytivirus, blocked viral replication. Furthermore, whole-genome transcriptome analysis revealed that ORF75 knockdown altered the transcription of 43 viral genes. Taken together, these results indicate that ORF75 encoded a highly protective immunogen that is also a global transcription regulator of Megalocytivirus.

  • P247 and P523: Two In Vivo-Expressed Megalocytivirus Proteins That Induce Protective Immunity and Are Essential to Viral Infection
    PloS one, 2015
    Co-Authors: Jian Zhang, Bao-cun Zhang, Li Sun
    Abstract:

    Megalocytivirus is a DNA virus with a broad host range among teleost fish. Although the complete genome sequences of a number of Megalocytivirus isolates have been reported, the functions of most of the genes of this virus are unknown. In this study, we selected two Megalocytivirus immunogens, P247 and P523, which were expressed during host infection and, when in the form of DNA vaccines (pCN247 and pCN523 respectively), elicited strong protectivity against lethal Megalocytivirus challenge in a turbot (Scophthalmus maximus) model. Compared to control fish, fish vaccinated with pCN247 and pCN523 exhibited drastically reduced viral loads in tissues and high levels of survival rates. Immune response analysis showed that pCN247 and pCN523 (i) induced production of specific serum antibodies, (ii) caused generation of cytotoxic immune cells and specific memory immune cells that responded to secondary antigen stimulation, and (iii) upregulated the expression of genes involved in innate and adaptive immunity. To examine the potential role of P247 and P523 in viral infection, the expression of P247 and P523 was knocked down by siRNA. Subsequent in vivo infection study showed that P247 and P523 knockdown significantly impaired viral replication. Furthermore, whole-genome transcriptome analysis revealed that P247 and P523 knockdown altered the expression profiles of 26 and 41 viral genes, respectively, putatively participating in diverse aspects of viral infection. Taken together, these results indicate that P247 and P523 induce protective immunity in teleost and play fundamental roles essential to viral replication. These observations provide the first evidence that suggests a likely link between the protectivity of viral immunogens and their biological significance in viral replication.

  • Effect of P247 and P523 knockdown on viral gene expression on a global scale.
    2015
    Co-Authors: Jian Zhang, Bao-cun Zhang, Li Sun
    Abstract:

    Turbot administered with psiP247 (A), psiP523 (B), or psiCR (control) were infected with Megalocytivirus, and the expression of 119 viral genes in the spleen was determined by quantitative real time RT-PCR.

  • Survival of vaccinated fish.
    2015
    Co-Authors: Jian Zhang, Bao-cun Zhang, Li Sun
    Abstract:

    Turbot vaccinated with pCN3, pCN247, pCN523, and PBS (Control) were challenged with Megalocytivirus and monitored daily for survival. Significance between the survivals of the vaccinated fish and the control fish was determined with logrank test. **P < 0.01.