Tiger Frog Virus Research Articles

Emerging threat of ranavirus: prevalence, genetic diversity, and climatic drivers of Ranavirus (Iridoviridae) in ectothermic vertebrates of Asia

IntroductionRanavirus disease, caused by viruses within the genus Ranavirus (Iridoviridae), is considered a globally emerging infectious disease linked to mass mortality events in both wild and cultured ectothermic vertebrates. Surveillance work is, however, limited in Asia hence prevalence and the dynamics of the disease remain poorly understood. To understand disease burden and the potential biotic and abiotic drivers in southern China region, we conducted a systematic surveillance of the ranavirus across Guangxi Zhuang Autonomous region (GAR).MethodsFor this, we used a multifaceted approach involving screening of amphibians and other potential hosts, diagnostic tests, phylogenetic analyses, prevalence estimation, co-infection assessments, and climatic niche analyses. Over one thousand individuals were sampled across 25 sampling sites.ResultsWe found ninety-two individuals from 18 species of ectothermic vertebrates to be infected with ranavirus. Two lineages were responsible – Rana nigromaculata ranavirus and tiger frog virus were identified using phylogenetic analysis based on the major capsid protein (MCP) gene fragment. Out of these two lineages, the presence of tiger frog virus is rare as we came across only one case. We also found evidence of a co-infection with ranavirus and Batrachochytrium dendrobatidis that can be highly detrimental to host populations; possibly the first such documentation in Asia. Our niche modelling analysis suggests that precipitation seasonality plays an important role in ranavirus prevalence in GAR – southwestern, southeastern, central and northeastern regions of GAR can be considered to be optimum habitats for ranaviruses. Infection rates in wild frog species have reached 100% in some areas, even in nature reserves.DiscussionOur research also indicates that culture facilities and pet markets are frequently infected, serving as likely vectors for the regional and global spread of ranaviruses. The knowledge generated suggests the need for systematic surveillance, stringent biosecurity measures, and control of international animal trade to prevent further transmission and protection of biodiversity and aquaculture industries across Asia.

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.

Molecular Confirmation of Ranavirus Infection in Amphibians From Chad, Africa.

Ranaviruses are DNA viruses (Family Iridoviridae; Subfamily Alphairidovirinae) and ranaviral disease is considered an emerging infectious disease of ectothermic vertebrates. Ranavirus infection can have varying pathological effects on infected amphibians, reptiles, and fish, most notably causing significant mortality events and population declines. Despite having a broad global range with reports from six continents, only a single incidental finding in Xenopus longipes from mainland Africa (Cameroon) is known and lacks molecular confirmation. Thus, there is a considerable knowledge gap concerning ranaviruses in Africa. We opportunistically obtained tissue samples from 160 amphibians representing five genera (Hoplobatrachus, Hylarana, Ptychadena, Pyxicephalus, and Xenopus) and two turtles (Pelomedusa sp.) from Chad, Africa. Samples were tested for ranavirus infection using a conventional PCR assay targeting the major capsid protein (MCP). A total of 25/160 (16%) frogs tested positive including 15/87 (17%) Hoplobatrachus occipitalis, 10/58 (17%) Ptychadena spp., 0/3 Pyxicephalus spp., 0/9 Xenopus spp., and 0/3 Hylarana spp. One of two turtles tested positive. Partial MCP gene sequences indicated all samples were >98% similar to several frog virus 3 (FV3)-like sequences. Additional gene targets (DNA polymerase [DNApol], ribonucleotide reductase alpha [RNR- α], ribonucleotide reductase beta subunit [RNR- β]) were sequenced to provide further detailed classification of the virus. Sequences of individual gene targets indicate that the ranavirus detected in frogs in Chad is most similar to tiger frog virus (TFV), a FV3-like virus previously isolated from diseased amphibians cultured in China and Thailand. Full genome sequencing of one sample indicates that the Chad frog virus (CFV) is a well-supported sister group to the TFVs previously determined from Asia. This work represents the first molecular confirmation of ranaviruses from Africa and is a first step in comparing ranavirus phylogeography on a local and global scale.

Roles of extracellular matrix components in Tiger frog virus attachment to fathead minnow (Pimephales promelas) cells

Tiger frog virus (TFV) belongs to the genus Ranavirus (family Iridoviridae) and causes significant harm in cultured frogs, resulting in substantial losses in ecological and economic field in Southern China. Attachment is the first step in viral life cycle, which is dependent on the interactions of virions with extracellular matrix (ECM) components. Studying this process will help in understanding virus infection and controlling viral diseases. In this study, the roles of primary ECM components in TFV attachment were investigated. The results on the kinetics of virus attachment showed TFV successful attachment to the cell surface as a relatively rapid process after TFV was used to inoculate cells for 10 min at 4 °C. Western blot and quantitative PCR analyses results showed that soluble fibronectin, collagen IV, laminin, or hyaluronic acid treatment with TFV caused no significant effect on virus attachment. Soluble heparin, heparan sulfate and chondroitin sulfate A/B could inhibit TFV attachment in a dose-dependent manner. Enzymic digestion by cell surface heparin/heparan sulfate using heparinase I, II, and III could significantly prevent TFV attachment, suggesting that heparan sulfate plays an important role in TFV attachment. Furthermore, the binding assays of heparin–agarose beads and virion showed that TFV virions specifically bound with heparin in a dose-dependent manner. Given that heparin is a structural analogue of heparan sulfate, the above results suggest that heparan sulfate might serve as an attachment factor of TFV infection. Our work would be beneficial to understand the mechanisms of TFV attachment and the interactions of TFV with cellular receptor(s).

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.

Phylogenomic characterization of ranaviruses isolated from cultured fish and amphibians in Thailand

Ranaviruses are emerging pathogens associated with worldwide epizootics in farmed and wild ectothermic vertebrates. In this study, we determined the full genomes of eight ranaviruses isolated from marbled sleeper goby ( Oxyeleotris marmorata), goldfish ( Carassius auratus), guppy ( Poecilia reticulata), tiger frog ( Hoplobatrachus tigerinus), Asian grass frog ( Fejervarya limnocharis), and East Asian bullfrog ( H. rugulosus) cultured or imported into Thailand. These ranaviral isolates induced the same cytopathic effects (i.e., progression of coalescing round plaques) in epithelioma papulosum cyprini (EPC) cell cultures. Transmission electron microscopy of infected EPC cells revealed cytoplasmic viral particles with ultrastructural features typical for ranaviruses. Pairwise genetic comparisons of the complete major capsid protein coding sequences from the Thai ranaviruses displayed the highest identity (99.8%–100%) to a ranavirus (tiger frog virus; TFV) isolated from diseased tiger frogs cultured in China, a slightly lower identity (99.3%–99.4%) to a ranavirus (Wamena virus; WV) isolated from diseased green tree pythons ( Morelia viridis) illegally exported from Papua New Guinea, and a lower identity to 35 other ranaviruses (93.7%–98.6%). Phylogenomic analyses supported the eight Thai ranaviruses, Chinese TFV, and WV as a subclade within a larger frog virus 3 clade. Our findings confirm the spread of TFV among cultured fish and amphibians in Asia and likely in reptiles in Oceania. Biosecurity measures are needed to ensure TFV does not continue to spread throughout Southeast Asia and to other parts of the world via international trade.

Tiger frog virus ORF104R interacts with cellular VDAC2 to inhibit cell apoptosis

Ranaviruses belong to the family Iridoviridae, and have become a serious threat to both farmed and natural populations of fish and amphibians. Previous reports showed that ranaviruses could encode viral Bcl-2 family-like proteins (vBcl-2), which play a critical role in the regulation of cell apoptosis. However, the mechanism of ranaviruses vBcl-2 interactions with host protein in mediating apoptosis remains unknown. Tiger frog virus (TFV) belonging to the genus Ranavirus has been isolated from infected tadpoles of Rana tigrina rugulosa, and it causes a high mortality rate among tiger frog tadpoles cultured in southern China. This study elucidated the molecular mechanism underlying the interaction of TFV ORF104R with the VDAC2 protein to regulate cell apoptosis. TFV ORF104R is highly similar to other ranaviruses vBcl-2 and host Mcl-1 proteins, indicating that TFV ORF104R is a postulate vBcl-2 protein. Transcription and protein expression levels showed that TFV orf104r was a late viral gene. Western blot results suggested that TFV ORF104R was a viral structural protein. Subcellular localization analysis indicated that TFV ORF104R was predominantly colocalized with the mitochondria. Overexpressed TFV ORF104R could suppress the release of cytochrome C and the activities of caspase-9 and caspase-3. These results indicated that TFV ORF104R might play an important role in anti-apoptosis. Furthermore, the interaction between TFV ORF104R and VDAC2 was detected by co-immunoprecipitation in vitro. The above observations suggest that the molecular mechanism of TFV-regulated anti-apoptosis is through the interaction of TFV ORF104R with the VDAC2 protein. Our study provided a mechanistic basis for the ranaviruses vBcl-2-mediated inhibition of apoptosis and improved the understanding on how TFV subverts host defense mechanisms in vivo.

Budding of Tiger Frog Virus (an Iridovirus) from HepG2 Cells via Three Ways Recruits the ESCRT Pathway.

The cellular endosomal sorting complex required for transport (ESCRT) pathway is a multifunctional pathway involved in cell physiological activities. While the majority of RNA viruses bearing L-domains are known to hijack the ESCRT pathway to complete the budding process, the budding of large and complex enveloped DNA viruses, especially iridoviruses, has been rarely investigated. In the present study, we use the tiger frog virus (TFV) as a model to investigate whether iridoviruses are released from host cells through the ESCRT pathway. Inhibition of class E proteins and auxiliary proteins (VPS4A, VPS4B, Tsg101, Alix, and Nedd4.1) reduces extracellular virion production, which preliminarily indicates that the ESCRT pathway is involved in TFV release. The respective interactions of TFV VP031L, VP065L, VP093L with Alix, Tsg101, Nedd4 suggest the underlying molecular mechanism by which TFV gets access to the ESCRT pathway. Co-depletion of Alix, Tsg101, and Nedd4.1 induces a significant reduction in extracellular virion production, which implies the functional redundancy of host factors in TFV budding. Those results are first observation that iridovirus gains access to ESCRT pathway through three ways of interactions between viral proteins and host proteins. Our study provides a better understanding of the budding mechanism of enveloped DNA viruses.

Identification and differential expression analysis of MicroRNAs encoded by Tiger Frog Virus in cross-species infection in vitro.

BackgroundTiger frog virus (TFV), dsDNA virus of the genus Ranavirus and family Iridoviridae, causes a high mortality of tiger frog tadpoles cultured in Southern China. MicroRNAs (miRNAs) have been identified in many viruses especially DNA viruses such as Singapore Grouper Iridoviruses (SGIV). MicroRNAs play important roles in regulating gene expression for virus subsistence in host. Considering that TFV infects cells of different species under laboratory conditions, we aim to identify the specific and essential miRNAs expressed in ZF4 and HepG2 cells.MethodsWe identified and predicted novel viral miRNAs in TFV-infected ZF4 and HepG2 cells by deep sequencing and software prediction. Then, we verified and described the expression patterns of TFV-encoded miRNAs by using qRT-PCR and Northern blot.ResultsDeep sequencing predicted 24 novel TFV-encoded miRNAs, and qRT-PCR verified 19 and 23 miRNAs in TFV-infected ZF4 (Group Z) and HepG2 (Group H) cells, respectively. Northern blot was performed to validate eight and five TFV-encoded miRNAs in Groups H and Z, respectively. We compared the expression of TFV-encoded miRNAs from two groups and defined TFV-miR-11 as the essential viral miRNA and TFV-miR-13 and TFV-miR-14 as the specific miRNAs that contribute to HepG2 cell infection.ConclusionsWe identified novel viral miRNAs and compared their expression in two host cells. The results of this study provide novel insights into the role of viral miRNAs in cross-species infection in vitro.Electronic supplementary materialThe online version of this article (doi:10.1186/s12985-016-0530-6) contains supplementary material, which is available to authorized users.

Tiger frog virus ORF080L protein interacts with LITAF and impairs EGF-induced EGFR degradation

Tiger frog virus (TFV) belongs to the genus Ranavirus, family Iridoviridae, and causes severe mortality in commercial cultures in China. TFV ORF080L is a gene homolog of lipopolysaccharide-induced TNF-α factor (LITAF), which is a regulator in endosome-to-lysosome trafficking through its function in the endosomal sorting complex required for transport machinery. The characteristics and biological roles of TFV ORF080L were identified. TFV ORF080L was predicted to encode an 84-amino acid peptide (VP080L). It had high-sequence identity with mammalian LITAF, but lacked the N-terminus of LITAF, which contains two PPXY motifs. Transcription and protein level analyses showed that TFV ORF080L was a late viral gene. Localization in the virons also showed that TFV VP080L was a viral structural protein. Immunofluorescence staining showed that TFV ORF080L was predominantly colocalized with plasma membrane and partly distributed with the late endosome in infected HepG2 cells. SiRNA-mediated TFV ORF080L silencing decreased viral reproduction. Moreover, TFV ORF080L interacted with human/zebrafish LITAF and impaired EGF-induced EGFR degradation, thereby indicating that TFV ORF080L played a role in endosome-to-lysosome trafficking. These findings suggested that TFV ORF080L might negate the function of cellular LITAF to impair endosomal sorting and trafficking. Results provide a clue to the link between the dysregulated endosomal trafficking and iridovirus pathogenesis.

Caveolae Restrict Tiger Frog Virus Release in HepG2 cells and Caveolae-Associated Proteins Incorporated into Virus Particles.

Caveolae are flask-shaped invaginations of the plasma membrane. Caveolae play important roles in the process of viruses entry into host cells, but the roles of caveolae at the late stage of virus infection were not completely understood. Tiger frog virus (TFV) has been isolated from the diseased tadpoles of the frog, Rana tigrina rugulosa, and causes high mortality of tiger frog tadpoles cultured in Southern China. In the present study, the roles of caveolae at the late stage of TFV infection were investigated. We showed that TFV virions were localized with the caveolae at the late stage of infection in HepG2 cells. Disruption of caveolae by methyl-β-cyclodextrin/nystatin or knockdown of caveolin-1 significantly increase the release of TFV. Moreover, the interaction between caveolin-1 and TFV major capsid protein was detected by co-immunoprecipitation. Those results suggested that caveolae restricted TFV release from the HepG2 cells. Caveolae-associated proteins (caveolin-1, caveolin-2, cavin-1, and cavin-2) were selectively incorporated into TFV virions. Different combinations of proteolytic and/or detergent treatments with virions showed that caveolae-associated proteins were located in viral capsid of TFV virons. Taken together, caveolae might be a restriction factor that affects virus release and caveolae-associated proteins were incorporated in TFV virions.

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.

Genome-wide analyses of proliferation-important genes of Iridovirus-tiger frog virus by RNAi

Tiger frog virus (TFV), a species of genus Ranavirus in the family Iridoviridae, is a nuclear cytoplasmic large DNA virus that infects aquatic vertebrates such as tiger frog (Rana tigrina rugulosa) and Chinese soft-shelled turtle (Trionyx sinensis). Based on the available genome sequences of TFV, the well-developed RNA interference (RNAi) technique, and the reliable cell line for infection model, we decided to analyze the functional importance of all predicted genes. Firstly, a relative quantitative cytopathogenic effect (Q-CPE) assay was established to monitor the viral proliferation in fish cells. Then, genome-wide RNAi screens of 95 small interference (si) RNAs against TFV were performed to characterize the functional importance of nearly all (95%) predicted TFV genes by Q-CPE scaling system. We identified 32 (33.7%) genes as essential, 50 (52.6%) genes as semi-essential and 13 (13.7%) genes as nonessential for TFV proliferation. Quantitative RT-PCR and titer assays of selected genes were performed to verify the screen results. Furthermore, the screened essential genes were analyzed for their genome distribution and conservative comparison within Ranavirus. Such a systematic screen for viral functional genes by cell phenotypes should provide further insights into understanding of the information in antiviral targets, and in viral replication and pathogenesis of iridovirus.

Iridovirus Infection in Chinese Giant Salamanders, China, 2010

Iridovirus Infection in Chinese Giant Salamanders, China, 2010

Entry of Tiger Frog Virus (an Iridovirus) into HepG2 Cells via a pH-Dependent, Atypical, Caveola-Mediated Endocytosis Pathway

Tiger frog virus (TFV), in the genus Ranavirus of the family Iridoviridae, causes high mortality of cultured tiger frog tadpoles in China. To explore the cellular entry mechanism of TFV, HepG2 cells were treated with drugs that inhibit the main endocytic pathways. We observed that TFV entry was inhibited by NH(4)Cl, chloroquine, and bafilomycin, which can all elevate the pH of acidic organelles. In contrast, TFV entry was not influenced by chlorpromazine or overexpression of a dominant-negative form of Esp15, which inhibit the assembly of clathrin-coated pits. These results suggested that TFV entry was not associated with clathrin-mediated endocytosis, but was related to the pH of acidic organelles. Subsequently, we found that endocytosis of TFV was dependent on membrane cholesterol and was inhibited by the caveolin-1 scaffolding domain peptide. Dynamin and actin were also required for TFV entry. In addition, TFV virions colocalized with the cholera toxin subunit B, indicating that TFV enters as caveola-internalized cargo into the Golgi complex. Taken together, our results demonstrated that TFV entry occurs by caveola-mediated endocytosis with a pH-dependent step. This atypical caveola-mediated endocytosis is different from the clathrin-mediated endocytosis of frog virus 3 (FV3) by BHK cells, which has been recognized as a model for iridoviruses. Thus, our work may help further the understanding of the initial steps of iridovirus infection in lower vertebrates.

Cloning, characterization, and biological function analysis of the SidT2 gene from Siniperca chuatsi

The systemic RNA interference defective protein (SID)-1 plays an important role in dsRNA uptake in cells. We identified the ScSidT2 gene from the mandarin fish ( Siniperca chuatsi), which is the first-studied SID-1 homolog in fish. ScSidT2 mRNA is 3380 bp long and contains a 2568-bp open reading frame that encodes an 855-amino-acid protein with an N-terminal signal peptide and ten putative transmembrane domains. Tissue distribution profile in healthy fish and expression profiles of ScSidT2 in infectious spleen and kidney necrosis virus (ISKNV)-infected fish were analyzed. Overexpression of the ScSidT2 protein in fathead minnow (FHM) epithelial cells could remarkably increase the uptake of exogenous dsRNA. In tiger frog virus (TFV)-infected FHM cells, overexpression of ScSidT2 could suppress virus production, and this mechanism could be significantly enhanced by adding virus-specific dsRNA. In mandarin fish fry cells, ISKNV infection and reproduction could be promoted when the ScSidT2 protein was neutralized with specific antisera. These results suggest that ScSidT2 could be associated with the host's antiviral defense mechanism.

Development and evaluation of a loop-mediated isothermal amplification assay for rapid detection of lymphocystis disease virus

A loop-mediated isothermal amplification (LAMP) assay was developed for the detection of lymphocystis disease virus (LCDV). A set of five specific primers, two inner and two outer primers and a loop primer, were designed on the basis of the major capsid protein gene of LCDV. The reaction time and temperatures were optimized for 60 min at 63 °C, respectively. LAMP amplification products were detected by a ladder-like appearance on agarose gel electrophoresis or a naked-eye inspection of a color change in the reaction tube by addition of SYBR Green I. The assay was specific for LCDV, and there was no cross-reactivity with white spot syndrome virus (WSSV) or six other Iridoviridae viruses (epizootic hematopoietic necrosis virus, EHNV; tiger frog virus, TFV; Bohle iridovirus, BIV; soft-shelled turtle iridovirus, STIV; infectious spleen and kidney necrosis virus, ISKNV; red sea bream iridovirus, RSIV). The detection limit of the LAMP assay was 15 fg, which was similar to that of real-time quantitative polymerase chain reaction (PCR) and 10-fold higher than the conventional PCR. The LAMP assay was evaluated using 109 clinical samples, and the results indicated the suitability and simplicity of the test as a rapid, field diagnostic tool for detection of LCDV. The LCDV LAMP assay has potential for early diagnosis of LCDV infection.

Complete sequence determination of a novel reptile iridovirus isolated from soft-shelled turtle and evolutionary analysis of Iridoviridae

BackgroundSoft-shelled turtle iridovirus (STIV) is the causative agent of severe systemic diseases in cultured soft-shelled turtles (Trionyx sinensis). To our knowledge, the only molecular information available on STIV mainly concerns the highly conserved STIV major capsid protein. The complete sequence of the STIV genome is not yet available. Therefore, determining the genome sequence of STIV and providing a detailed bioinformatic analysis of its genome content and evolution status will facilitate further understanding of the taxonomic elements of STIV and the molecular mechanisms of reptile iridovirus pathogenesis.ResultsWe determined the complete nucleotide sequence of the STIV genome using 454 Life Science sequencing technology. The STIV genome is 105 890 bp in length with a base composition of 55.1% G+C. Computer assisted analysis revealed that the STIV genome contains 105 potential open reading frames (ORFs), which encode polypeptides ranging from 40 to 1,294 amino acids and 20 microRNA candidates. Among the putative proteins, 20 share homology with the ancestral proteins of the nuclear and cytoplasmic large DNA viruses (NCLDVs). Comparative genomic analysis showed that STIV has the highest degree of sequence conservation and a colinear arrangement of genes with frog virus 3 (FV3), followed by Tiger frog virus (TFV), Ambystoma tigrinum virus (ATV), Singapore grouper iridovirus (SGIV), Grouper iridovirus (GIV) and other iridovirus isolates. Phylogenetic analysis based on conserved core genes and complete genome sequence of STIV with other virus genomes was performed. Moreover, analysis of the gene gain-and-loss events in the family Iridoviridae suggested that the genes encoded by iridoviruses have evolved for favoring adaptation to different natural host species.ConclusionThis study has provided the complete genome sequence of STIV. Phylogenetic analysis suggested that STIV and FV3 are strains of the same viral species belonging to the Ranavirus genus in the Iridoviridae family. Given virus-host co-evolution and the phylogenetic relationship among vertebrates from fish to reptiles, we propose that iridovirus might transmit between reptiles and amphibians and that STIV and FV3 are strains of the same viral species in the Ranavirus genus.

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 α-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.

Development of an RT-PCR detection method for mud crab reovirus

Mud crab reovirus (MCRV) causes high mortality in the cultivated mud crab. To control better an outbreak of this virus, a rapid, specific and sensitive detection method based on RT-PCR was developed. The MCRV detection method was designed based on the one-step and two-step RT-PCR which resulted in the amplification of predicted products of 433 and 304 bp. The method is specific as no cross-reaction was observed between grass carp hemorrhage virus (GCHV), white spot syndrome virus (WSSV), tiger frog virus (TFV), infectious spleen and kidney necrosis virus (ISKNV), fish nervous necrosis virus (NNV) and the muscovy duck virus (MDRV). One-step PCR amplification could detect 10 −8 μg of purified MCRV dsRNA, while two-step PCR amplification could detect 10 −9 μg MCRV dsRNA. At an early stage of infection, MCRV could be detected in the heart, thoracic ganglion, muscle, intestine, gut, hemolymph, gonad and gill, but not in the stomach or hepatopancreas. However, in the moribund mud crabs, MCRV could be detected in all tissues examined.