Chuanfu Dong

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.

Characterization of a new cell line from caudal fin of koi, Cyprinus carpio koi, and first isolation of cyprinid herpesvirus 3 in China

A new continuous cell line (KCF-1) from caudal fin of koi, Cyprinus carpio koi, was developed and sub-cultured more than 100 passages since the present study was initiated in March 2006. KCF-1 predominantly consisted of short fibroblast-like cells and grew well in Dulbeccos modified Eagle medium (DMEM) supplemented with 10% fetal bovine serum (FBS). Chromosome analysis revealed that 56% of the KCF-1 cells maintained normal diploid chromosome number (2n=100) at Passage 82. Using the KCF-1 cell line, a strain of cyprinid herpesvirus 3 (designated as CyHV-3-QY08) was isolated from the diseased koi. CyHV-3-QY08 continuously propagated in the KCF-1 cells, as confirmed by immunofluorescence assay (IFA) and transmission electron microscopy (TEM). KCF-1 cells infected with CyHV-3-QY08 produced typical cytopathic effects characterized by severe vacuolation, deformation of nuclei, and marginalization of the nuclear chromatin, which are consistent with those of previous reports. CyHV-3-QY08 was purified and subsequently analyzed by SDS-PAGE and TEM. The results showed that the purified virions contained two types of morphologies and were composed of more than 30 obvious viral polypeptides. An infectivity experiment revealed that CyHV-3-QY08 could cause 100% mortality in the infected koi. Based on the genome sequence of CyHV-3-I/U, the CyHV-3(I/U)-ORF136 homologue in CyHV-3-QY08 was cloned and sequenced. Multiple sequence alignments of CyHV-3-I/U-ORF136 homologues showed that CyHV-3-QY08 belonged to the typical Asian genotype. The CyHV-3(I/U)-ORF136 homologue seems to be a novel molecule marker, which can be used to distinguish Asia isolates from Europe-America strains.

Proteomic analysis of zebrafish (Danio rerio) infected with infectious spleen and kidney necrosis virus

Iridovirus infections remain a severe problem in aquaculture industries worldwide. Infectious spleen and kidney necrosis virus (ISKNV), the type species of the genus Megalocytovirus in the family Iridoviridae, has caused significant economic losses among freshwater fish in different Asian countries. To investigate the molecular mechanism of iridoviral pathogenesis, we analyzed the differential proteome from the spleen of ISKNV-infected zebrafish through two-dimensional gel electrophoresis (2-DE). Mass spectrometry revealed 35 altered cellular protein spots, including 15 upregulated proteins and 20 downregulated proteins at five days post-infection. The altered host proteins were classified into 13 categories based on their biological processes: cytoskeletal protein, stress response, lipoprotein metabolism, ubiquitin-proteasome pathway, carbohydrate metabolism, signal transduction, proteolysis, ion binding, transport, metabolic process, catabolic process, biosynthesis, and oxidation reduction. Moreover, 14 corresponding genes of the differentially expressed proteins were validated by RT-PCR. Western blot analysis further demonstrated the changes in α-tubulin, β-actin, HSC70, and major capsid protein (MCP) during infection. β-Actin was selected for further study via co-immunoprecipitation analyses, which confirmed that the cellular β-actin interacts with the MCP protein of ISKNV in the infected zebrafish. These findings provide insight into the interactions between iridoviruses (especially ISKNV) and host, as well as the mechanism and pathogenesis of ISKNV infections.

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.

Tiger frog virus can infect zebrafish cells for studying up- or down-regulated genes by proteomics approach

Tiger frog virus (TFV), a member of the iridovirus family, causes high mortality of cultured tiger frog tadpoles in southern China. To better understand TFV infection and its interaction with host cells, zebrafish embryonic fibroblast (ZF4) cells, a stable polyploid cell line with most clear genetic map, was used for our present study. Our results showed that TFV caused typical lytic plaque forming cytopathic effect (CPE) and that various stages of viral proliferation were observed using electron microscopy and indirect immunofluorescence assay. Two-dimensional electrophoresis also showed that some cellular proteins were differentially expressed in the ZF4 cells infected with TFV. A total of 10 proteins were identified using the matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) technique, including 7 that were up-regulated and 3 that were down-regulated after infection. Among the 10 identified proteins, alterations in Hsp90 and alpha-tubulin expression were further confirmed by Western blot analysis. Furthermore, reorganization of microtubules was also observed in TFV-infected cells and can probably be attributed to the overexpression of translationally controlled tumor protein. The present study is the first attempt to reveal cellular responses to TFV infection by proteomics. The results suggest that the ZF4 cell line could be used as a model to study TFV infection.

Emergence of fatal European genotype CyHV-3/KHV in mainland China.

Cyprinid herpesvirus 3 (CyHV-3), also known as koi herpesvirus (KHV), is a highly infectious causative agent to common carp and koi worldwide. The virus is mainly consisted of European and Asian genotype isolates. To date, no European genotype CyHV-3 has been found emerging in the East and Southeast Asian regions. In late March 2011, an outbreak of CyHV-3 disease occurred in Guangzhou City, Guangdong Province, China, resulting in the deaths of approximately 200 large-sized adult koi within four weeks. One moribund koi was sampled for CyHV-3 isolation. Thus, a CyHV-3 was isolated in KCF-1 cells and designated as KHV-GZ11. Abundant mature or immature virions in infected KCF-1 cells were observed under a transmission electron micrograph. In addition, intra-nuclear inclusion body-like structures with masses of virions were also observed. Based on the TK and ORF136H genes, the sequence analyses revealed that KHV-GZ11 is a distinct European genotype of CyHV-3. Moreover, the infectivity experiment showed that KHV-GZ11 was highly virulent to koi. In summary, we are the first to confirm the emergence of fatal European genotype CyHV-3/KHV in East and Southeast Asia. Our study will provide new insight to explore the virus origin and epidemiology, as well as its pathogenicity.

Global landscape of structural proteins of infectious spleen and kidney necrosis virus.

ABSTRACT Infectious spleen and kidney necrosis virus (ISKNV), the type species of the genus Megalocytivirus in the family Iridoviridae, causes severe damage to mandarin fish cultures in China. Little is known about the proteins of ISKNV virions. In this study, a total of 38 ISKNV virion-associated proteins were identified by four different workflows with systematic and comprehensive proteomic approaches. Among the 38 identified proteins, 21 proteins were identified by the gel-based workflows (one-dimensional [1-D] and two-dimensional [2-D] gel electrophoresis). Fifteen proteins were identified by 1-D gel electrophoresis, and 16 proteins were identified by 2-D gel electrophoresis, with 10 proteins identified by both methods. Another 17 proteins were identified only by liquid chromatography (LC)-based workflows (LC-matrix-assisted laser desorption ionization [MALDI] and linear trap quadrupole [LTQ]-Orbitrap). Among these 17 LC-identified proteins, 5 proteins were identified uniquely by the LC-MALDI workflow, whereas another 6 proteins were identified only by the LTQ-Orbitrap workflow. These results underscore the importance of incorporation of multiple approaches in identification of viral proteins. Based on viral genomic sequence, genes encoding these 38 viral proteins were cloned and expressed in vitro. Antibodies were produced against these 38 proteins to confirm the ISKNV structural proteins by Western blotting. Of the newly identified proteins, ORF 056L and ORF 118L were identified and confirmed as two novel viral envelope proteins by Western blotting and immunoelectron microscopy (IEM). The ISKNV proteome reported here is currently the only characterized megalocytivirus proteome. The systematic and comprehensive identification of ISKNV structural proteins and their localizations in this study will facilitate future studies of the ISKNV assembly process and infection mechanism.

Characteristics of the interferon regulatory factor pairs zfIRF5/7 and their stimulation expression by ISKNV Infection in zebrafish (Danio rerio).

The interferon regulatory factor (IRF) family plays critical roles in a hosts virus infection responses. In this study, two IRF family members, zfIRF5 and zfIRF7, are identified in zebrafish. The zfIRF5 protein encodes 297 amino acids without the carboxyl IRF3 domain. We suggest that zfIRF5 is a new kind of splicing variant, following the nine other kinds of IRF5 splicing variants found in mammals. The zfIRF7 protein is identified as a member of the IRF7 family, compared to the human IRF7 protein, the amino acid sequence of zfIRF7 only with 29% identity and devoid a virus activated domain (VAD). There zfIRF5/7 proteins are expressed in all 11 selective zebrafish tissues within 6-120h of embryonic development. Laser confocal microscopy shows that the full length the proteins are separately located in the cytoplasm. Mutation experiments show that the nuclear localization signals (NLS) of zfIRF7 and zfIRF-5 are at the N-terminal and C-terminals, respectively. In the assays, zfIRF7 expression increases during infectious spleen and kidney necrosis virus (ISKNV) infection and by poly(I:C) and LPS injections, both of which activate the transcriptional activity of L8G5-luc plasmids. The over-expression of zfIRF5/7 activates the interferon-stimulated response elements (ISRE) signal pathway. In addition, zfIRF7 can activate IFN-β, zfIRF5/7. Co-immunoprecipitation assays and laser co-confocal microscopy show that the two proteins could interact, and zfIRF7 may stimulate zfIRF5 to move into the nucleus. The co-expression of zfIRF5/IRF7 suppresses the transcriptional activities of IFN-β in HEK293T cells.

Characterization of a TnMAVS protein from Tetraodon nigroviridis

A growing family of cellular proteins encoding for caspase activation and the recruitment domain (CARD) plays a crucial role in immunity by sensing viral infections and signaling antiviral immune defenses. We obtained a MAVS-like protein (named TnMAVS) from Tetradon nigroviridis, which contains a CARD domain, a pro-rich domain, and a TM domain similar to human MAVS. A fluorescence assay showed that TnMAVS was located in the cytoplasm and near by the membrane, and not the mitochondria in FHM cells. As such, it was considered as a new member of MAVS. The TnMAVS was highly expressed in the liver and muscle of T. nigroviridis. In the spleen, TnMAVS was down-regulated when the fish was treated with polyinosinic:polycytidylic acid or challenged with ISKNV, but was not affected by PGN or LPS. The dual luciferase reporter assay revealed that TnMAVS overexpression resulted in the activation of the interferon-sensitive response element and NF-κB signal pathways. In addition, a characteristic TRAF3-associated peptide PVQD was found in the TnMAVS sequence. Co-immunoprecipitation assays indicated that TnMAVS could interact with zfTRAF3 in eukaryotic cells.

Extracellular virion proteins of two Chinese CyHV-3/KHV isolates, and identification of two novel envelope proteins

Proteins in extracellular virions of two Chinese cyprinid herpesvirus-3/koi herpesvirus (CyHV-3/KHV) isolates were identified through one-dimensional gel-based matrix-assisted laser desorption/ionization tandem time of flight mass spectrometry in combination with liquid chromatography tandem mass spectrometry (LC ESI-MS/MS). A total of 43 viral proteins were identified, seven of which (pORF62, 68, 43, 51, 92, 84, and 72) were characterized as marked characteristic viral proteins in CyHV-3/KHV virions and six of which (pORF11, 27, 83, 91, 106 and 116) were not detected previously. Of the newly identified proteins, pORF83 was identified as a novel viral minor envelope protein by Western blot analysis and indirect immunofluorescence assay. Another 27 cellular proteins were validated by LC ESI-MS/MS to be involved in purified extracellular CyHV-3/KHV virions, among which a highly abundant, virus-inducible stress protein was shown and characterized as a cell-derived envelope protein in CyHV-3/KHV virions. Our study extends the number of known CyHV-3/KHV virion-associated viral proteins from 40 to 46 and identifies a novel viral envelope protein as well as a cellular envelope protein. The present findings provide some new insights for better understanding CyHV-3/KHV.