Critical Role of an MHC Class I-Like/Innate-Like T Cell Immune Surveillance System in Host Defense against Ranavirus (Frog Virus 3) Infection.

Besides the central role of classical Major Histocompatibility Complex (MHC) class Ia-restricted conventional Cluster of Differentiation 8 (CD8) T cells in antiviral host immune response, the amphibian Xenopuslaevis critically rely on MHC class I-like (mhc1b10.1.L or XNC10)-restricted innate-like (i)T cells (iVα6 T cells) to control infection by the ranavirus Frog virus 3 (FV3). To complement and extend our previous reverse genetic studies showing that iVα6 T cells are required for tadpole survival, as well as for timely and effective adult viral clearance, we examined the conditions and kinetics of iVα6 T cell response against FV3. Using a FV3 knock-out (KO) growth-defective mutant, we found that upregulation of the XNC10 restricting class I-like gene and the rapid recruitment of iVα6 T cells depend on detectable viral replication and productive FV3 infection. In addition, by in vivo depletion with XNC10 tetramers, we demonstrated the direct antiviral effector function of iVα6 T cells. Notably, the transitory iV6 T cell defect delayed innate interferon and cytokine gene response, resulting in long-lasting negative inability to control FV3 infection. These findings suggest that in Xenopus and likely other amphibians, an immune surveillance system based on the early activation of iT cells by non-polymorphic MHC class-I like molecules is important for efficient antiviral immune response.

Evaluating the Importance of Environmental Persistence for Ranavirus Transmission and Epidemiology.

Viruses persist outside their hosts in a variety of forms, from naked virions to virus protected in sloughed tissues or carcasses, and for a range of times, all of which affect the likelihood and importance of transmission from the environment. This review synthesizes the literature on environmental persistence of viruses in the genus Ranavirus (family Iridoviridae), which are large double-stranded DNA viruses of ectothermic, often aquatic or semiaquatic vertebrates. Ranaviruses have been associated with mass mortality events in natural and captive settings around the world, and with population and community-wide declines in Europe. Early work suggested ranaviruses are environmentally robust and transmission from the environment should be common. More recent work has shown a large effect of temperature and microbial action on persistence times, although other aspects of the environment (e.g., water chemistry) and aquatic communities (e.g., zooplankton) may also be important. Ranaviruses may persist in the carcasses of animals that have died of infection, and so decomposing organisms and invertebrate scavengers may reduce these persistence times. The question is, do persistence times vary enough to promote or preclude substantial transmission from the environment. We built an epidemiological model with transmission from contacts, free virus in water, and carcasses, to explore the conditions in which environmental persistence could be important for ranavirus epidemiology. Based on prior work, we expected a substantial amount of transmission from the water and that longer persistence times would make this route of transmission dominant. However, neither water-borne nor transmission from carcasses played an important role in the simulated epidemics except under fairly restrictive conditions, such as when there were high rates of virus shedding or high rates of scavenging on highly infectious carcasses. While many aspects of environmental persistence of ranaviruses are being resolved by experiments, key parameters such as viral shedding rates are virtually unknown and will need to be empirically constrained if we are to determine whether environmental persistence and transmission from the environment are essential or insignificant features of Ranavirus epidemiology. We conclude by emphasizing the need to place environmental persistence research in an epidemiological framework.

A quantitative-PCR based method to estimate ranavirus viral load following normalisation by reference to an ultraconserved vertebrate target.

Ranaviruses are important pathogens of amphibians, reptiles and fish. To meet the need for an analytical method for generating normalised and comparable infection data for these diverse host species, two standard-curve based quantitative-PCR (qPCR) assays were developed enabling viral load estimation across these host groups. A viral qPCR targeting the major capsid protein (MCP) gene was developed which was specific to amphibian-associated ranaviruses with high analytical sensitivity (lower limit of detection: 4.23 plasmid standard copies per reaction) and high reproducibility across a wide dynamic range (coefficient of variation below 3.82% from 3 to 3×108 standard copies per reaction). The comparative sensitivity of the viral qPCR was 100% (n=78) based on agreement with an established end-point PCR. Comparative specificity with the end-point PCR was also 100% (n=94) using samples from sites with no history of ranavirus infection. To normalise viral quantities, a host qPCR was developed which targeted a single-copy, ultra-conserved non-coding element (UCNE) of vertebrates. Viral and host qPCRs were applied to track ranavirus growth in culture. The two assays offer a robust approach to viral load estimation and the host qPCR can be paired with assays targeting other pathogens to study infection burdens.

Fish miR-146a promotes Singapore grouper iridovirus infection by regulating cell apoptosis and NF-κB activation.

miR-146a was reported to participate in various pathophysiological conditions in mammals, such as inflammation and immune responses, oncogenesis and cell damage. However, its function in low vertebrates has not been well elucidated. In this study, we characterized the expression profiles and functions of miR-146a in fish cells during iridovirus infection. We found that the reported fish miR-146a genes encoded an identical mature sequence, which shared high similarity with its mammalian orthologues, suggesting a putative functional conservation of miR-146a between fish and other vertebrates. Using a well-established infection model of Singapore grouper iridovirus (SGIV) in fathead minnow cells, we found that SGIV infection induced the expression of miR-146a to a dramatic extent. More importantly, we found that miR-146a promoted SGIV propagation, as demonstrated by higher expression of viral genes and increased virus titres in miR-146a-overexpressing cells. Mechanistically, we found that miR-146a overexpression suppressed, while miR-146a knockdown promoted, NF-κB activation and SGIV-induced cell apoptosis, two major cellular events involved in SGIV infection. Our study suggested that the induction of miR-146a by SGIV infection may function through a feed-forward mechanism to promote viral infection by restraining anti-viral cellular responses.

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

BACKGROUND: Tiger 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. METHODS: We 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. RESULTS: Deep 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. CONCLUSIONS: We 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.

Quantitative study of proteomic alterations in a Zebrafish (danio rerio) cell line infected with the Singapore Grouper Iridovirus (SGIV).

We demonstrate, for the first time, that Singapore Grouper Iridovirus (SGIV) can successfully infect a Zebrafish cell line. Combined with the recent availability of the complete zebrafish (danio rerio) genome, this provides an opportunity to investigate virus-host interactions at the molecular level. Using iTRAQ labeling and two-dimensional LC/MS/MS quantitative proteomics, 157 zebrafish proteins exhibiting significant alterations in expression levels following SGIV infection were identified. Gene ontology analysis revealed that SGIV controls a wide aspect of zebrafish host machinery to ensure replication and propagation. In order to probe the mechanism underlying SGIV infection in Zebrafish cells, we used an anti-sense morpholino to knockdown orf86r, an immediate early viral gene that encodes the SGIV protein ORF86R. The expression profile of certain host proteins involved in replication was altered upon knockdown. In particular, expression of CNOT, a non-enzymatic subunit of the CCR4-NOT transcription complex was markedly affected. Taken together, these findings provide a new insight on the function of the essential viral protein ORF86R. Our results show that Singapore Grouper Iridovirus infection of a Zebrafish cell line is a useful new tool to study virus-host interactions.

Clinical signs, pathology and dose-dependent survival of adult wood frogs, Rana sylvatica, inoculated orally with frog virus 3 Ranavirus sp., Iridoviridae.

Amphibian populations suffer massive mortalities from infection with frog virus 3 FV3, genus Ranavirus, family Iridoviridae, a pathogen also involved in mortalities of fish and reptiles. Experimental oral infection with FV3 in captive-raised adult wood frogs, Rana sylvatica Lithobates sylvaticus, was performed as the first step in establishing a native North American animal model of ranaviral disease to study pathogenesis and host response. Oral dosing was successful LD50 was 10(2.93 2.423.44) p.f.u. for frogs averaging 35mm in length. Onset of clinical signs occurred 614days post-infection p.i. median 11 days p.i. and time to death was 1014 days p.i. median 12 days p.i.. Each tenfold increase in virus dose increased the odds of dying by 23-fold and accelerated onset of clinical signs and death by approximately 15. Ranavirus DNA was demonstrated in skin and liver of all frogs that died or were euthanized because of severe clinical signs. Shedding of virus occurred in faeces 710 days p.i. 34.5days before death and skin sheds 10 days p.i. 01.5days before death of some frogs dead from infection. Most common lesions were dermal erosion and haemorrhages haematopoietic necrosis in bone marrow, kidney, spleen and liver and necrosis in renal glomeruli, tongue, gastrointestinal tract and urinary bladder mucosa. Presence of ranavirus in lesions was confirmed by immunohistochemistry. Intracytoplasmic inclusion bodies probably viral were present in the bone marrow and the epithelia of the oral cavity, gastrointestinal tract, renal tubules and urinary bladder. Our work describes a ranaviruswood frog model and provides estimates that can be incorporated into ranavirus disease ecology models.

Development of an Ussuri catfish Pseudobagrus ussuriensis skin cell line displaying differential cytopathic effects to three aquatic animal viruses.

An Ussuri catfish Pseudobagrus ussuriensis skin (UCS) cell line was developed and subcultured for more than 60 passages. UCS cells consisted of mostly epithelial-like cells and multiplied well in TC199 medium supplemented with 10% fetal bovine serum at 25°C. Chromosome analysis revealed that most UCS cells had a normal diploid karyotype with 2n=52. UCS cells showed differential cytopathic effects (CPEs) after inoculation of spring viremia of carp virus (SVCV, a negative-strand RNA virus), grass carp reovirus (GCRV, a multi-segmented double-stranded RNA virus) and Rana grylio virus (RGV, a large double-stranded DNA virus), and were indicative of high sensitivities to these three aquatic animal viruses by a virus titration study. The CPE caused by SVCV appeared as rounded and granular cells, grape-like clusters and small lytic plaques. Characteristic CPE containing plaque-like syncytia was induced by GCRV. RGV-infected cells produced typical CPE characterized by cells shrinkage and aggregation, formation of clear plaques and cell sheet detachment. Furthermore, significant fluorescent signals were observed after UCS cells were transfected with green fluorescent protein reporter plasmids, and the development of CPE induced by a recombinant RGV, ΔTK-RGV, in UCS cells was illustrated using a combination of light and fluorescence microscopy. The data from this study suggested that UCS cell line can potentially serve as a useful tool for the comparison study of different aquatic animal viruses and the isolation of some newly emerging viruses in Ussuri catfish farming.

Susceptibility of Xenopus laevis tadpoles to infection by the ranavirus Frog-Virus 3 correlates with a reduced and delayed innate immune response in comparison with adult frogs.

Xenopus laevis adults mount effective immune responses to ranavirus Frog Virus 3 (FV3) infections and clear the pathogen within 2-3 weeks. In contrast, most tadpoles cannot clear FV3 and succumb to infections within a month. While larval susceptibility has been attributed to ineffective adaptive immunity, the contribution of innate immune components has not been addressed. Accordingly, we performed a comprehensive gene expression analysis on FV3-infected tadpoles and adults. In comparison to adults, leukocytes and tissues of infected tadpoles exhibited modest (10-100 time lower than adult) and delayed (3 day later than adult) increase in expression of inflammation-associated (TNF-α, IL-1ß and IFN-γ) and antiviral (Mx1) genes. In contrast, these genes were readily and robustly upregulated in tadpoles upon bacterial stimulation. Furthermore, greater proportions of larval than adult PLs were infected by FV3. Our study suggests that tadpole susceptibility to FV3 infection is partially due to poor virus-elicited innate immune responses.

Differential viral propagation and induction of apoptosis by grouper iridovirus (GIV) in cell lines from three non-host species.

Grouper iridovirus (GIV), belonging to the Ranavirus genus of the Iridoviridae family, was demonstrated to differentially express viral genes and induce apoptosis in three non-host fish cell lines rainbow trout monocyte/macrophage (RTS11), chinook salmon embryonic (CHSE-214) and fathead minnow Epithelioma papulosum cyprinid (EPC). These cells were challenged with GIV and virus entry into all three cell lines was confirmed by the expression of viral immediate early genes. The expression of the late major capsid protein gene was detected in CHSE-214 and EPC, but not in RTS11, suggesting an earlier termination in the viral replication cycle in RTS11. Approximately 12h after infection with GIV, cell death was prominent in all three non-host cell lines. Death was later confirmed to be apoptosis by the presence of chromosomal DNA fragmentation and phosphatidylserine externalization. To determine whether apoptosis was protein related or gene expression related, the three cell lines were challenged with heat-inactivated GIV and UV-treated GIV (GIV(UV)). The heat inactivation abolished apoptosis in all three cell lines, but each cell line responded differently to GIV(UV). Relative to GIV, GIV(UV) caused no apoptosis in CHSE-214, decreased apoptosis in RTS11, and increased apoptosis in EPC. These results suggest that early GIV gene expression was needed for apoptosis in CHSE-214 but impeded apoptosis in EPC. At the cellular level, only EPC is a permissive host as EPC was the only cell line of the three capable of producing a moderate increase in virus titer. The three non-host cell lines present a good system for potentially identifying different components of GIV-induced apoptotic pathways in future studies.

Rana grylio virus as a vector for foreign gene expression in fish cells.

In the present study, Rana grylio virus (RGV, an iridovirus) thymidine kinase (TK) gene and viral envelope protein 53R gene were chosen as targets for foreign gene insertion. ΔTK-RGV and Δ53R-RGV, two recombinant RGV, expressing enhanced green fluorescence protein (EGFP) were constructed and analyzed in Epithelioma papulosum cyprinid (EPC) cells. The EGFP gene which fused to the virus major capsid protein (MCP) promoter p50 was inserted into TK and 53R gene loci of RGV, respectively. Cells infected with these two recombinant viruses not only displayed plaques, but also emitted strong green fluorescence under fluorescence microscope, providing a simple method for selection and purification of recombinant viruses. ΔTK-RGV was purified by seven successive rounds of plaque isolation and could be stably propagated in EPC cells. All of the plaques produced by the purified recombinant virus emitted green fluorescence. However, Δ53R-RGV was hard to be purified even through twenty rounds of plaque isolation. The purified recombinant virus ΔTK-RGV was verified by PCR analysis and Western blotting. These results showed EGFP was expressed in ΔTK-RGV infected cells. Furthermore, one-step growth curves and electron microscopy revealed that infection with recombinant ΔTK-RGV and wild-type RGV are similar. Therefore, RGV was demonstrated could be as a viral vector for foreign gene expression in fish cells.

Improved knockout methodology reveals that frog virus 3 mutants lacking either the 18K immediate-early gene or the truncated vIF-2alpha gene are defective for replication and growth in vivo.

To better assess the roles of frog virus 3 (FV3; genus Ranavirus, family Iridoviridae) genes in virulence and immune evasion, we have developed a reliable and efficient method to systematically knock out (KO) putative virulence genes by site-specific integration into the FV3 genome. Our approach utilizes a dual selection marker consisting of the puromycin resistance gene fused in frame with the enhanced green fluorescent protein (EGFP) reporter (Puro-EGFP cassette) under the control of the FV3 immediate-early (IE) 18K promoter. By successive rounds of selection for puromycin resistance and GFP expression, we have successfully constructed three recombinant viruses. In one, a "knock-in" mutant was created by inserting the Puro-EGFP cassette into a noncoding region of the FV3 genome (FV3-Puro/GFP). In the remaining two, KO mutants were constructed by replacement of the truncated viral homolog of eIF-2α (FV3-ΔvIF-2α) or the 18K IE gene (FV3-Δ18K) with the Puro-EGFP cassette. The specificity of recombination and the clonality of each mutant were confirmed by PCR, sequencing, and immunofluorescence microscopy. Viral replication of each recombinant in cell culture was similar to that of parental FV3; however, infection in Xenopus laevis tadpoles revealed that FV3-ΔvIF-2α and FV3-Δ18K replicated less and resulted in lower mortality than did GFP-FV3 and wild-type FV3. Our results suggest that 18K, which is conserved in all ranaviruses, and the truncated vIF-2α gene contribute to virulence. In addition, our study describes a powerful methodology that lays the foundation for the discovery of potentially new ranaviral genes involved in virulence and immune escape.

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.

Inactivation of frog virus 3 and channel catfish virus by esculentin-2P and ranatuerin-2P, two antimicrobial peptides isolated from frog skin.

While it is clear that some amphibian populations have recently experienced precipitous declines, the causes of those die-offs are complex and likely involve multiple variables. One theory suggests that environmental factors may trigger events that result in depressed immune function and increased susceptibility to infectious disease. Here we examine one aspect of innate immunity in amphibians and show that esculentin-2P (E2P) and ranatuerin-2P (R2P), two antimicrobial peptides isolated from Rana pipiens, inactivate frog virus 3, a potentially pathogenic iridovirus infecting anurans, and channel catfish herpesvirus. In contrast to mammalian antimicrobial peptides, E2P and R2P act within minutes, at temperatures as low as 0 degrees C, to inhibit viral infectivity. Moreover, these compounds appear to inactivate the virus directly and do not act by inhibiting replication in infected cells. This is the first report linking amphibian antimicrobial peptides with protection from an amphibian viral pathogen and suggests that these compounds may play a role in maintaining amphibian health.

Instability of frog virus 3 mRNA in productively infected cells.

Cloned DNA restriction fragments encoding representative frog virus 3 messages were used as probes to assess the stability of viral transcripts in infected fathead minnow cells. Analysis of Northern blot hybridization profiles confirmed earlier findings and revealed that in infected cells the steady-state level of representative frog virus 3 (FV3) messages increased throughout the replication cycle. However, when actinomycin D was added at 4 hr after infection to block the synthesis of new transcripts, viral messages were observed to turn over rapidly, with half-lives of approximately 2 hr. These results indicate that viral transcripts were not preferentially stabilized in FV3-infected cells and suggest that the high steady-state level of viral messages present at late times after infection was due to viral transcription outpacing message degradation. Moreover, the instability of viral messages challenges the suggestion that the terminal dyad symmetry (hairpin structure) observed in all frog virus 3 messages sequenced to date plays a role in transcript stability.