Soft-shelled Turtle Iridovirus Research Articles

First Report of Endemic Frog Virus 3 (FV3)-like Ranaviruses in the Korean Clawed Salamander (Onychodactylus koreanus) in Asia.

Frog virus 3 (FV3) in the genus Ranavirus of the family Iridoviridae causes mass mortality in both anurans and urodeles worldwide; however, the phylogenetic origin of FV3-like ranaviruses is not well established. In Asia, three FV3-like ranaviruses have been reported in farmed populations of amphibians and reptiles. Here, we report the first case of endemic FV3-like ranavirus infections in the Korean clawed salamander Onychodactylus koreanus, caught in wild mountain streams in the Republic of Korea (ROK), through whole-genome sequencing and phylogenetic analysis. Two isolated FV3-like ranaviruses (Onychodactylus koreanus ranavirus, OKRV1 and 2) showed high similarity with the Rana grylio virus (RGV, 91.5%) and Rana nigromaculata ranavirus (RNRV, 92.2%) but relatively low similarity with the soft-shelled turtle iridovirus (STIV, 84.2%) in open reading frame (ORF) comparisons. OKRV1 and 2 formed a monophyletic clade with previously known Asian FV3-like ranaviruses, a sister group of the New World FV3-like ranavirus clade. Our results suggest that OKRV1 and 2 are FV3-like ranaviruses endemic to the ROK, and RGV and RNRV might also be endemic strains in China, unlike previous speculation. Our data have great implications for the study of the phylogeny and spreading routes of FV3-like ranaviruses and suggest the need for additional detection and analysis of FV3-like ranaviruses in wild populations in Asian countries.

Transcriptiomics analysis reveals immue regulation and cell death regulation of IFNg in Pelodiscus sinensis STA cells

Our previous study showed that overexpression interferon gamma (IFNg) in soft-shelled turtle (Pelodiscus sinensis) artery cell line (STA cells) could induce soft-shelled turtle iridovirus (STIV)-infected cell death and inhibit STIV replication. To understand the molecular basis, four samples (named as Ctrl, Ctrl_STIV, IFNg and IFNg_STIV) including cells treatment with recombinant IFNg (rIFNg) or not, and infected or mock-infected with STIV were used to transcriptome analysis. A total of 77.18 Gb clean data were obtained and the clean data of each sample was over 5.97 Gb. Compared to the Ctrl group, there are 2172, 1192 and 2638 DEGs were detected, with 1127, 580 and 1349 up-regulated genes and 1045, 612, and 1259 down-regulated genes, in STA cells after treatment with IFNg, Ctrl_STIV and IFNg_STIV respectively. Subsequently, DEGs were annotated and enriched to GO items and the KEGG pathway, the result showed that immune activation of rIFNg in STA cells including Jak-STAT signaling activation, pattern recognition receptor (PRR) mediated innate immunity activation and cell death regulation etc. The activation of PRR mediated innate immunity may contribute to antiviral activity of IFNg, and it was not affected by STIV infection. From the aspect of cell death, rIFNg could regulated a series of cell death associated Go items including positive regulation and negative regulation, however some GO items including negative regulation of cell death were no longer enriched after STIV infection. The enriched death-related KEGG pathways including “Apoptosis” and “Necroptosis” were enriched in STA cells after rIFNg treatment, and however, besides “Necroptosis”, “Cellular senescence”, “p53 signaling pathway” were enriched after STIV infection. All of these indicate that rIFNg could build an antiviral activity in STA cells and may induce STIV-infected cell death through limiting the negative regulation of cell death and inducing necroptosis, apoptosis and cellular senescence.

Gene synteny, evolution and antiviral activity of type I IFNs in a reptile species, the Chinese soft-shelled turtle Pelodiscus sinensis

Type I interferons (IFNs) are critical cytokines for the establishment of antiviral status in fish, amphibian, avian and mammal, but the knowledge on type I IFNs is rather limited in reptile. In this study, seven type I IFN genes, designed as IFN1 to IFN7, were identified from a reptile species, the Chinese soft-shelled turtle (Pelodiscus sinensis). These identified type I IFNs have relatively low protein identity, when compared with those in human and chicken; but they possess conserved cysteines, predicted multi-helix structure and N-terminal signal peptide. The Chinese soft-shelled turtle IFN1 to IFN5 have two exons and one intron, but IFN6 and IFN7 are the single-exon genes. Chinese soft-shelled turtle type I IFNs are located respectively on the two conserved reptile-bird loci, named as Locus a and Locus c, and are clustered into the four of the five reptile-bird groups (named as Groups I–V) based on phylogenetic evidence, due to the lack of IFNK in the turtle. Moreover, the Chinese soft-shelled turtle type I IFNs can be induced by soft-shelled turtle iridovirus (STIV) infection and show antiviral activity in soft-shelled turtle artery (STA) cells, except IFN6. In addition, due to the difference in genome organizations, such as the number of exons and introns of type I IFN genes from fish to mammal, the definition and evolution of ‘intronless’ type I IFN genes were discussed in lineages of vertebrates. Thus, the finding of type I IFNs on two different loci in P. sinensis sheds light on the evolution of type I IFN genes in vertebrates.

Onsite and visual detection of ranavirus in largemouth bass (Micropterus salmoides) by an isothermal recombinase polymerase amplification method

Largemouth bass ranavirus (LMBV) has caused mortality in largemouth bass and led to huge economic losses in the aquaculture industry. Here, we developed a novel, fast, and simple isothermal recombinase polymerase amplification assay (RPA) for LMBV detection with primers designed on the basis of a fragment of the major capsid protein gene. The reaction conditions were optimized and the RPA method was specific for LMBV, as the DNA of other four Iridoviridae viruses (Singapore grouper iridovirus, soft-shelled turtle iridovirus, tiger frog virus, and large yellow croaker iridovirus), white spot syndrome virus, grass carp reovirus, and healthy largemouth bass could not be amplified. The detection limit of LMBV-RPA was 89 copies/μL, which was comparable to the sensitivity determined by real-time PCR. Finally, the RPA method was validated to be a simple, convenient, rapid, and field diagnostic tool for LMBV detection using 10 clinical samples. In this paper, the RPA method is firstly applied to the diagnosis and monitoring of LMBV infection in the aquaculture industry of largemouth bass, which shows great prospects for on-site diagnostics of LMBV using nearly free instruments.

Generating aptamers for specific recognition against soft-shelled turtle iridovirus infection

Soft-shelled turtle iridovirus (STIV) causes devastating virus disease in farmed soft-shelled turtles (Trionyx sinensis). Aptamers with remarkably high specificity and affinity have been widely applied in diseases diagnosis and therapies. In this study, single-stranded DNA (ssDNA) aptamers were generated against STIV-infected Fathead minnow (FHM) cells via Cell-SELEX (Cell based-Systematic Evolution of Ligands by Exponential enrichment technology). Aptamers could form stable stem-loop structures and specifically recognize STIV-infected FHM cells, with calculated dissociation constants (Kd) of 191 ± 12 and 497 ± 39 nM for aptamers LYST1and LYST2, respectively, which indicated that they could serve as specific delivery system for antiviral therapies. Both aptamers displayed antiviral activities in vitro. The targets of aptamers LYST1and LYST2 in cell membranes could be membrane proteins, which were trypsin-sensitive. FAM-labeled aptamer LYST1 could identify STIV infection in diseased turtle tissues. It is the first time that aptamers against STIV-infected cells have been selected and characterized. The aptamers have great potential in development of rapid diagnostic assays and targeted antiviral drugs against STIV in aquaculture.

Rapid diagnosis of largemouth bass ranavirus in fish samples using the loop-mediated isothermal amplification method

Largemouth bass ranavirus (LMBV) has been recognized as the causative pathogen responsible for infectious skin ulcerative syndrome in cultured largemouth bass in China. A fast and simple LMBV detection method is urgently needed. Here, a loop-mediated isothermal amplification (LAMP) assay was established for the detection of this virus using primers targeting the major capsid protein gene of LMBV. The amplification conditions were optimized; the assay was specific for the diagnosis of LMBV, as there was no cross-reactivity with other four Iridoviridae viruses (large yellow croaker iridovirus, Singapore grouper iridovirus, tiger frog virus, and soft-shelled turtle iridovirus), grass carp reovirus, white spot syndrome virus, or healthy largemouth bass. The sensitivity of the LAMP assay was found to be 8.55 × 101 copies/μL of LMBV DNA, which was 10-fold higher than that of the conventional PCR. Application of the LAMP assay was evaluated using 10 clinical samples, and the results indicated the reliability of the test as a rapid, field diagnostic tool for LMBV detection. Thus, the simplicity and nearly instrument-free LAMP method provides an alternative for rapid and sensitive detection of LMBV and has great potential for early diagnosis of LMBV infection in the farm.

Characterization and functional analysis of liver-expressed antimicrobial peptide-2 (LEAP-2) in Pelodiscus sinensis

Liver-expressed antimicrobial peptide-2 (LEAP-2) is a cationic antimicrobial peptide with two disulfide bonds, and it has been shown to play an important role in the host defence in fish, amphibians, birds and mammals. Here, we characterize LEAP-2 from Pelodiscus sinensis and identify its function. The deduced precursor of PsLEAP-2 was composed of 77 amino acids (aa), including a 22-aa signal peptide, a 15-aa pro-domain with a typical cleavage site (R34XXR37), and a 40-aa mature peptide with four conserved cysteine residues that form two disulfide bonds (Cys54, 60, 65, 70). PsLEAP-2 was highly conserved in evolution, and the precursor/mature peptide of PsLEAP-2 showed 42%–62%/57%–72% sequence identity with LEAP-2 sequences from other known tetrapods. Expression analysis data showed that PsLEAP-2 was only expressed in the liver. RT-qPCR results showed that the mRNA expression level of PsLEAP-2 was induced after challenge with Edwardsiella tarda (E. tarda) or soft-shelled turtle iridovirus (STIV). In freshly isolated liver cells, induced expression of PsLEAP-2 was only observed at 24 h after stimulation with LPS or poly(I:C). Epithelioma papulosum cyprinid (EPC) cells transfected with the LEAP-2-Flag plasmid exhibited a reduced intake of E. tarda and reduced viral yield of STIV compared with cells transfected with the Flag plasmid. These results suggest that PsLEAP-2 plays an important role in the immune defence against bacterial and viral infection.

Soft-shelled turtle iridovirus enters cells via cholesterol-dependent, clathrin-mediated endocytosis as well as macropinocytosis.

Ranaviruses are nucleoplasmic large DNA viruses that can cause major economic losses in the aquaculture industry and pose a severe threat to global ecological diversity. The available literature demonstrates that classifiable members of the genus Ranavirus enter cells via multiple and complicated routes. Here, we demonstrated the underlying cellular entry mechanism of soft-shelled turtle iridovirus (STIV) using green fluorescence tagged recombinant virus. Treatment with chlorpromazine, sucrose, ethyl-isopropyl amiloride, chloroquine or bafilomycin A1 all significantly decreased STIV infection, suggesting that STIV uses clathrin-mediated endocytosis and macropinocytosis to enter cells via a pH-dependent pathway. Depletion of cellular cholesterol with methyl-β-cyclodextrin significantly inhibited STIV entry, but neither filipin III nor nystatin did, suggesting that STIV entry was cholesterol dependent but caveola independent. Treatment with dynasore, genistein, ML-7 or cytochalasin D all significantly inhibited STIV infection, indicating that Rac GTPase and myosin II activity were required for the macropinocytosis-like pathway as well as actin polymerization. Our findings suggest that the molecular events involved in STIV entry are not identical to those of other ranavirus isolates. Our results also extend our understanding of the molecular mechanism of iridovirus entry and pathogenesis.

Characterization of DNA aptamers generated against the soft-shelled turtle iridovirus with antiviral effects.

BackgroundSoft-shelled turtle iridovirus (STIV) causes severe systemic disease in farmed soft-shelled turtles (Trionyx sinensis). More efficient methods of controlling and detecting STIV infections are urgently needed. MethodsIn this study, we generated eight single-stranded DNA (ssDNA) aptamers against STIV using systematic evolution of ligands by exponential enrichment (SELEX).ResultsThe aptamers formed representative stem-loop secondary structures. Electrophoretic mobility shift assays and fluorescent localization showed that the selected aptamers had high binding affinity for STIV. Aptamer QA-36 had the highest calculated binding affinity (Kd) of 53.8 nM. Flow cytometry and fluorescence microscopy of cell-aptamer interactions demonstrated that QA-12 was able to recognize both STIV-infected cells and tissues with a high level of specificity. Moreover, the selected aptamers inhibited STIV infection in vitro and in vivo, with aptamer QA-36 demonstrating the greatest protective effect against STIV and inhibiting STIV infection in a dose-dependent manner.DiscussionWe generated DNA aptamers that bound STIV with a high level of specificity, providing an alternative means for investigating STIV pathogenesis, drug development, and medical therapies for STIV infection.ConclusionsThese DNA aptamers may thus be suitable antiviral candidates for the control of STIV infections.

Development of a new cell line from the snout of giant grouper, Epinephelus lanceolatus (Bloch), and its application in iridovirus and nodavirus pathogenesis

We developed and characterized a new marine fish cell line (ELGSN) from the snout of giant grouper, Epinephelus lanceolatus (Bloch). Two different phenotypes of cells, ELGSN(e), which consisted predominantly of epithelial-like cells, and ELGSN(f), which consisted predominantly of fibroblast-like cells, were isolated and cultured. Both ELGSN, and ELGSN(f) cells were subcultured for >80 passages and multiplied well in Leibovitz's L-15 medium supplemented with 10% fetal bovine serum at 28 degrees C. Karyotyping analysis indicated that the modal chromosome numbers of ELGSN(e) and ELGSN(f) cells were 48 and 62, respectively. After transfection with EGFP-C1, bright green fluorescence was observed in these two cells and the transfection efficiency was 35% and 28%, respectively. Moreover, the two types of ELGSN cells both showed susceptibility to Singapore grouper iridovirus (SGIV) and red-spotted grouper nervous necrosis virus (RGNNV), but not to soft-shelled turtle iridovirus (STIV) and spring viremia of carp virus (SVCV), indicated by the occurrence of severe cytopathic effect and increased viral titers. In SGIV-infected cells, paracrystalline arrays, different stages of virus particles and amorphous structures were observed under electron microscopy. In RGNNV-infected cells, numerous virus particles and circular virus-packed macropinosome-like structures were observed. All these data suggested that ELGSN cells could be used for effective virus propagation in vitro. In addition, we provided biochemical evidences that macropinocytosis was involved in RGNNV infection in ELGSN cells. Taken together, the new established ELGSN cell lines provided a useful tool for transgenic and genetic manipulation, as well as virus propagation and pathogenesis. (C) 2014 Elsevier B.V. All rights reserved.

Selection and characterization of single-chain recombinant antibodies against infectious haematopoietic necrosis virus from mouse phage display library

Six single-chain fragment variable (scFv) antibodies against infectious haematopoietic necrosis virus (IHNV) were selected from an antibody phage display library by phage display technology. The soluble scFv antibodies showed a molecular weight 32kDa by Western blot. Dot blot analysis revealed that the six scFv antibodies could recognize IHNV. For enzyme linked immunosorbent assay (ELISA), four scFv antibodies (P1A4, P1A12, P1D5 and P3E2) showed cross-reactivity with spring viraemia of carp virus (SVCV). However, none of the six scFv antibodies had cross-reaction with Pike fry rhabdovirus (PFRV), Soft-shelled turtle iridovirus (STIV), viral haemorrhagic septicemia virus (VHSV), or viral nervous necrosis virus (VNNV). Indirect immunofluorescence results showed that all of these scFv antibodies reacted positively with virus in the IHNV-infected cells. These scFv antibodies will be useful in diagnostic test development and pathogenesis studies for IHNV.

Establishment of a new cell line from the heart of giant grouper, Epinephelus lanceolatus (Bloch), and its application in toxicology and virus susceptibility

A new marine fish cell line, derived from the heart of giant grouper, Epinephelus lanceolatus (Bloch), was established and characterized. The cell line was designated as ELGH and subcultured with more than 60 passages. The ELGH cells were mainly composed of fibroblast-like cells and multiplied well in Leibovitz's L-15 medium supplemented with 10% foetal bovine serum (FBS) at 28 °C. Chromosome analysis indicated that the modal chromosome number was 48. The fluorescent signals were detected in ELGH when transfected with green fluorescent protein reporter plasmids. The 50% cytotoxic concentration (CC50 ) of the extracellular products (ECPs) from Streptococcus iniae and Vibrio alginolyticus E333 on ELGH cells was 60.02 and 12.49 μg mL(-1), respectively. Moreover, the ELGH cells showed susceptibility to Singapore grouper iridovirus (SGIV), but not to soft-shelled turtle iridovirus (STIV), red-spotted grouper nervous necrosis virus (RGNNV) and spring viremia of carp virus (SVCV), which was demonstrated by the presence of a severe cytopathic effect (CPE) and increased viral titres. In addition, electron microscopy observation showed that abundant virus particles were present in the infected cells. Taken together, our data above provided the potential utility of ELGH cells for transgenic and genetic manipulation, as well as cytotoxicity testing and virus pathogenesis.

IFN-γ in turtle: Conservation in sequence and signalling and role in inhibiting iridovirus replication in Chinese soft-shelled turtle Pelodiscus sinensis

The IFN-γ gene was identified in a turtle, the Chinese soft-shelled turtle, Pelodiscus sinensis, with its genome consisting of 4 exons and 3 introns. The deduced amino acid sequence of this gene contains a signal peptide, an IFN-γ family signature motif 130IQRKAVNELFPT, an NLS motif 155KRKR and three potential N-glycosylation sites. As revealed by real-time quantitative PCR, the gene was constitutively expressed in all tested organs/tissues, with higher level observed in blood, intestine and thymus. An induced expression of IFN-γ at mRNA level was observed in peripheral blood leucocytes (PBLs) in response to in vitro stimulation of LPS and PolyI:C. The overexpression of IFN-γ in the Chinese soft-shelled turtle artery (STA) cell line resulted in the increase in the expression of transcriptional regulators, such as IRF1, IRF7 and STAT1, and antiviral genes, such as Mx, PKR, implying possibly the existence of a conserved signalling network and role for IFN-γ in the turtle. Furthermore, the infection of soft-shelled turtle iridovirus (STIV) in the cell line transfected with IFN-γ may cause the cell death as demonstrated with the elevated lactate dehydrogenase (LDH) level and cell mortality. However, the mechanism involved in the antiviral activity may require further investigation.

A loop-mediated isothermal amplification method for the detection of members of the genus Ranavirus

A loop-mediated isothermal amplification (LAMP) method was developed for detection of members of the genus Ranavirus. The optimum reaction mixture contained 2.5 μL of each inner primer, RV-FIP (20pmol/μL) and RV-BIP (20pmol/μL), 0.5 μL of each outer primer, RV-F3 (10pmol/μL) and RV-B3 (10pmol/μL), 1.25 μL of each loop primer, RV-LF (20pmol/μL) and RV-LB (20pmol/μL), 3.5 μL dNTP mix (10mM each), 8 μL MgSO4 (25mM), 1 μL of Bst DNA polymerase (8 U/mL, large fragment; New England Biolabs Inc., Beverly, MA, USA), 2.5 μL 10×supplied buffer, and 1 μL of template DNA in a final volume of 25 μL. The optimum reaction conditions were 63 °C for 60min. This LAMP method could detect Andrias davidianus iridovirus (ADIV), soft-shelled turtle iridovirus (STIV), and epizootic hematopoietic necrosis virus (EHNV), all of which belong to the genus Ranavirus, but it could not detect other viruses such as koi herpes virus (KHV), channel catfish virus (CCV), infectious spleen and kidney necrosis virus (ISKNV) and white spot syndrome virus (WSSV). The detection limit of the LAMP method was 100 copies of STIV DNA segment, and the sensitivity was 10 times higher than that of the polymerase chain reaction (PCR) assay. The results could be estimated visually by eye when calcein was added.

Characterization of a ranavirus isolated from the Chinese giant salamander (Andrias davidianus, Blanchard, 1871) in China

In this study, we confirmed that a novel ranavirus was related to mass die-offs of farmed Chinese giant salamanders (Andrias davidianus) in Leshan, Sichuan Province, China. The farmed Chinese giant salamanders presented typical clinical symptoms that included ecchymoses or swollen areas on the head and limbs, necrosis of the limbs, and skin ulceration. Bacteriological and pathologic examinations further proved the possibility of ranavirus infection of the Chinese giant salamanders. The virus was isolated via inoculation in the epithelioma papulosum cyprini (EPC) cells. The virus multiplied and caused cytopathogenic effects (CPE) at 25°C in the EPC cells and it was sensitive to chloroform treatment, trypsinase treatment, heating at 56°C for 30min, acidity (pH 3.0), and alkalinity (pH=10.0). Treatment with 5-bromouracil deoxyriboside (5-BrdU) inhibited viral replication, which suggested that the viral genome contained DNA. After the viral inoculation in the cell, samples were collected and made the ultrathin sections, and then the iridovirus-like particles could be observed in the cytoplasm and nuclei by electron microscopy. Using the isolated viral DNA as the template for PCR, we amplified, cloned, and analyzed target sequences in three different genomic regions, i.e., the major capsid protein (MCP), DNA polymerase, and neurofilament triplet H1-like protein (NF-H1). Subsequent multiple alignment and phylogenetic analysis showed that the newly isolated virus was closely related to KRV-1, soft-shelled turtle iridovirus, Rana grylio iridovirus, and FV3. In the pathogenicity tests, the challenged salamanders showed similar clinical symptoms to the natural infections. Our data suggested that the virus isolate was a novel member of the genus Ranavirus, family Iridoviridae. We tentatively named the virus as Chinese giant salamander virus (CGSV-L). CGSV-L was able to cause serious systemic disease and it was capable of killing 80% of Chinese giant salamanders (body weight=300.0±15.0g).

Establishment of a Cell Line from Chinese Soft-shelled Turtle Pelodiscus sinensis with the Practicability of Transfection and Viral Replication

continuous cell line derived from extracranial carotid artery of the Chinese soft-shelled turtle Pelodiscus sinensis was established and characterized. It was named as STA (soft-shelled turtle artery) cell line. The cells were muscle-cell like, and were stained with antibody against smooth muscle myofilament marker, a-smooth muscle actin. These cells had a normal diploid chromosome number of 2n = 66, and their 12S rRNA and 16S rRNA sequences shared 99% identity with ones of the turtle in database. The cell line can be cultured well in media DMEM/F12 or M199 supplemented with 10% FBS at 28 C. It was further demonstrated that the cells were transfected successfully with pTurbo plasmid, with a transfection efficiency being over 30%. The soft-shelled turtle iridovirus (STIV) propagated in the cell line, causing typical CPE with the formation of inclusion bodies. It is suggested that the established STA cell line provides a convenient platform for studying the pathogenesis of STIV and biological aspects of the turtle.

Iridovirus Infection in Chinese Giant Salamanders, China, 2010

Iridovirus Infection in Chinese Giant Salamanders, China, 2010

Establishment and characterization of a new cell line derived from kidney of grouper, Epinephelus akaara (Temminck & Schlegel), susceptible to Singapore grouper iridovirus (SGIV)

A marine fish cell line derived from the kidney of red-spotted grouper, Epinephelus akaara, designated as EAGK was established and characterized. The EAGK cells multiplied well in Leibovitz's L-15 medium containing 10% foetal bovine serum at 25 °C and have been subcultured for more than 90 passages. Karyotyping, chromosomal typing and ribosomal RNA (rRNA) genotyping analysis revealed that EAGK had a modal diploid chromosome number of 82 and was a fibroblast cell line originated from grouper. A severe cytopathic effect was observed in EAGK cells incubated with Singapore grouper iridovirus (SGIV), but not with soft-shelled turtle iridovirus, viral nervous necrosis virus or spring viraemia of carp virus. SGIV replication was further confirmed by immunofluorescence, electron microscopy and virus titre determination. Bright fluorescence was observed after transfection with fluorescent protein reporter plasmids, indicating that EAGK cells can be used to identify gene functions in vitro. In addition, the cell organelles including mitochondria and endoplasm reticulum changed and aggregated around virus factories after SGIV infection, suggested that the EAGK cell line could be an important tool for investigation of iridovirus-host interactions.

Construction of green fluorescent protein-tagged recombinant iridovirus to assess viral replication

Green fluorescent protein-tagged recombinant virus has been successfully applied to observing the infective dynamics and evaluating viral replication. Here, we identified soft-shelled turtle iridovirus (STIV) ORF55 as an envelope protein (VP55), and developed a recombinant STIV expressing an enhanced green fluorescent protein (EGFP) fused to VP55 (EGFP-STIV). Recombinant EGFP-STIV shared similar single-step growth curves and ultrastructural morphology with wild type STIV (wt-STIV). The green fluorescence distribution during EGFP-STIV infection was consistent with the intracellular distribution of VP55 which was mostly co-localized with virus assembly sites. Furthermore, EGFP-STIV could be used to evaluate viral replication conveniently under drug treatment, and the result showed that STIV replication was significantly inhibited after the addition of antioxidant pyrrolidine dithiocarbamate (PDTC). Thus, the EGFP-tagged recombinant iridovirus will not only be useful for further investigations on the viral replicative dynamics, but also provide an alternative simple strategy to screen for antiviral substances.

Involvement of the mitogen-activated protein kinase pathway in soft-shelled turtle iridovirus-induced apoptosis

Iridoviruses are large DNA viruses that infect invertebrates and poikilothermic vertebrates, and result in significant economic losses in aquaculture production, and drastic declines in amphibian populations. Soft-shelled turtle iridovirus (STIV) is the causative agent of severe systemic diseases in farm-raised soft-shelled turtles (Trionyx sinensis). In the present study, the mechanisms of STIV-induced cell death and the roles of the mitogen-activated protein kinase (MAPK) signaling pathway were investigated. STIV infection evoked typical apoptosis in fish cells, as demonstrated by the formation of apoptotic bodies, positive terminal deoxynucleotidyl transferase-mediated nicked-end labeling, and caspase-3 activation. The translocation of cytochrome c from mitochondria to cytoplasm, and caspase-9 activation suggested that a mitochondria-mediated pathway was involved in STIV-induced apoptosis. Moreover, MAPK pathways, including extracellular signal-regulated kinase (ERK), c-Jun N-terminal kinase (JNK) and p38 MAPK signaling were activated during STIV infection. Using specific inhibitors, we found that MAPK signaling molecules, including ERK, JNK and p38 MAPK, were important for virus release, whereas, only ERK and p38 MAPK were involved in STIV-induced apoptosis by modulating caspase-3 activity. Taken together, our findings shed light on the roles of the MAPK signaling pathway in iridovirus-induced apoptosis and virus replication, which provides new insights into understanding iridovirus-host interaction.