Genomic Sequencing of Ranavirus Isolates from a Three-Spined Stickleback (Gasterosteus aculeatus) and a Red-Legged Frog (Rana aurora).

Two ranavirus isolates were recovered during a wildlife disease investigation in California in 1996. Preliminary testing at the time of sample collection indicated that the two isolates were identical. Phylogenetic analysis of the full genomes of these two isolates revealed that they are a single strain of frog virus 3.

Cloning and characterization of antiviral cytotoxic T lymphocytes in channel catfish, Ictalurus punctatus.

To determine the role of piscine anti-viral cytotoxic cells, we analyzed the response of channel catfish to Ictalurid herpesvirus 1, commonly designated channel catfish virus (CCV). Peripheral blood leukocytes (PBL) from catfish immunized with MHC-matched, CCV-infected G14D cells (G14D-CCV) showed marked lysis of G14D-CCV but little to no lysis of uninfected allogenic (3B11) or syngeneic (G14D) cells. Expansion of effectors by in vitro culture in the presence of irradiated G14D-CCV cells generated cultures with enhanced cytotoxicity and often broader target range. Cytotoxic effectors expressed rearranged TCR genes, perforin, granzyme, and IFN-γ. Four clonal cytotoxic lines were developed and unique TCR gene rearrangements including γδ were detected. Furthermore, catfish CTL clones were either CD4+/CD8- or CD4-/CD8-. Two CTL lines showed markedly enhanced killing of G14D-CCV targets, while the other two lines displayed a broader target range. Collectively, catfish virus-specific CTL display unique features that illustrate the diversity of the ectothermic vertebrate immune response.

Molecular and Ecological Studies of a Virus Family (Iridoviridae) Infecting Invertebrates and Ectothermic Vertebrates.

Research involving viruses within the family Iridoviridae (generically designated iridovirids to distinguish members of the family Iridoviridae from members of the genus Iridovirus) has markedly increased in recent years [...].

Genomic sequence of a Bohle iridovirus strain isolated from a diseased boreal toad (Anaxyrus boreas boreas) in a North American aquarium.

Genomic sequence analysis of zoo ranavirus (ZRV) suggests it is a strain of Bohle iridovirus (BIV), a virus that was first detected in, and thought to be confined to, Australia. Furthermore, marked sequence similarity and genomic co-linearity among ZRV, BIV, and German gecko ranavirus (GGRV) are consistent with the view that all three are strains  of Frog virus 3, the type species of the genus Ranavirus, family Iridoviridae.

Ranavirus phylogenomics: Signatures of recombination and inversions among bullfrog ranaculture isolates.

Ranaviruses are emerging pathogens of fish, amphibians, and reptiles that threaten aquatic animal industries and wildlife worldwide. Our objective was to genetically characterize ranaviruses isolated during separate bullfrog Lithobates catesbeianus die-offs that occurred eight years apart on the same North American farm. The earlier outbreak was due to a highly pathogenic strain of common midwife toad virus (CMTV) previously known only from Europe and China. The later outbreak was due to a chimeric ranavirus that displayed a novel genome arrangement and a DNA backbone typical for Frog virus 3 (FV3) strains except for interspersed fragments acquired through recombination with the CMTV isolated earlier. Both bullfrog ranaviruses are more pathogenic than wild-type FV3 suggesting recombination may have resulted in the increased pathogenicity observed in the ranavirus isolated in the later outbreak. Our study underscores the role international trade in farmed bullfrogs may have played in the global dissemination of highly pathogenic ranaviruses.

Ranaviruses and other members of the family Iridoviridae: Their place in the virosphere.

Members of the family Iridoviridae, collectively referred to as iridovirids, are large, double-stranded DNA-containing viruses that infect invertebrates and cold-blooded (ectothermic) vertebrates. Infections in the former often lead to massive levels of virus replication resulting in iridescence of the infected animal and ultimately death. Among the latter, infections target a variety of organs and are capable of causing high levels of morbidity and mortality among commercially and ecologically important fish and amphibian species. The viral replication strategy has been elucidated primarily through the study of frog virus 3 (FV3) with additional input from other iridovirids of ecological or commercial importance. Replication occurs within both nuclear and cytoplasmic compartments and involves synthesis of genome length and concatemeric DNA, extensive methylation of the viral genome (among vertebrate viruses only), coordinate expression of three classes of viral gene products, and formation of icosahedral virions within cytoplasmic viral assembly sites. Phylogenetic analyses delineate five genera within the family and suggest that members of the families Iridoviridae, Ascoviridae, and Marseilleviridae compromise a monophyletic lineage in which ascoviruses are most closely related to invertebrate iridoviruses.

ICTV Virus Taxonomy Profile: Iridoviridae.

The Iridoviridae is a family of large, icosahedral viruses with double-stranded DNA genomes ranging in size from 103 to 220 kbp. Members of the subfamily Alphairidovirinae infect ectothermic vertebrates (bony fish, amphibians and reptiles), whereas members of the subfamily Betairidovirinae mainly infect insects and crustaceans. Infections can be either covert or patent, and in vertebrates they can lead to high levels of mortality among commercially and ecologically important fish and amphibians. This is a summary of the current International Committee on Taxonomy of Viruses (ICTV) Report on the taxonomy of the Iridoviridae, which is available at www.ictv.global/report/iridoviridae.

Characterization of Frog Virus 3 knockout mutants lacking putative virulence genes.

To identify ranavirus virulence genes, we engineered Frog Virus 3 (FV3) knockout (KO) mutants defective for a putative viral caspase activation and recruitment domain-containing (CARD) protein (Δ64R-FV3) and a ß-hydroxysteroid dehydrogenase homolog (Δ52L-FV3). Compared to wild type (WT) FV3, infection of Xenopus tadpoles with Δ64R- or Δ52L-FV3 resulted in significantly lower levels of mortality and viral replication. We further characterized these and two earlier KO mutants lacking the immediate-early18kDa protein (FV3-Δ18K) or the truncated viral homolog of eIF-2α (FV3-ΔvIF-2α). All KO mutants replicated as well as WT-FV3 in non-amphibian cell lines, whereas in Xenopus A6 kidney cells replication of ΔvCARD-, ΔvßHSD- and ΔvIF-2α-FV3 was markedly reduced. Furthermore, Δ64R- and ΔvIF-2α-FV3 were more sensitive to interferon than WT and Δ18-FV3. Notably, Δ64R-, Δ18K- and ΔvIF-2α- but not Δ52L-FV3 triggered more apoptosis than WT FV3. These data suggest that vCARD (64R) and vß-HSD (52L) genes contribute to viral pathogenesis.

Environmental dependency of amphibian-ranavirus genotypic interactions: evolutionary perspectives on infectious diseases.

The context-dependent investigations of host-pathogen genotypic interactions, where environmental factors are explicitly incorporated, allow the assessment of both coevolutionary history and contemporary ecological influences. Such a functional explanatory framework is particularly valuable for describing mortality trends and identifying drivers of disease risk more accurately. Using two common North American frog species (Lithobates pipiens and Lithobates sylvaticus) and three strains of frog virus 3 (FV3) at different temperatures, we conducted a laboratory experiment to investigate the influence of host species/genotype, ranavirus strains, temperature, and their interactions, in determining mortality and infection patterns. Our results revealed variability in host susceptibility and strain infectivity along with significant host-strain interactions, indicating that the outcome of an infection is dependent on the specific combination of host and virus genotypes. Moreover, we observed a strong influence of temperature on infection and mortality probabilities, revealing the potential for genotype-genotype-environment interactions to be responsible for unexpected mortality in this system. Our study thus suggests that amphibian hosts and ranavirus strains genetic characteristics should be considered in order to understand infection outcomes and that the investigation of coevolutionary mechanisms within a context-dependent framework provides a tool for the comprehensive understanding of disease dynamics.

Isolation of a Bohle-like iridovirus from boreal toads housed within a cosmopolitan aquarium collection.

A captive 'survival assurance' population of 56 endangered boreal toads Anaxyrus boreas boreas, housed within a cosmopolitan collection of amphibians originating from Southeast Asia and other locations, experienced high mortality (91%) in April to July 2010. Histological examination demonstrated lesions consistent with ranaviral disease, including multicentric necrosis of skin, kidney, liver, spleen, and hematopoietic tissue, vasculitis, and myriad basophilic intracytoplasmic inclusion bodies. Initial confirmation of ranavirus infection was made by Taqman real-time PCR analysis of a portion of the major capsid protein (MCP) gene and detection of iridovirus-like particles by transmission electron microscopy. Preliminary DNA sequence analysis of the MCP, DNA polymerase, and neurofilament protein (NFP) genes demonstrated highest identity with Bohle iridovirus (BIV). A virus, tentatively designated zoo ranavirus (ZRV), was subsequently isolated, and viral protein profiles, restriction fragment length polymorphism analysis, and next generation DNA sequencing were performed. Comparison of a concatenated set of 4 ZRV genes, for which BIV sequence data are available, with sequence data from representative ranaviruses confirmed that ZRV was most similar to BIV. This is the first report of a BIV-like agent outside of Australia. However, it is not clear whether ZRV is a novel North American variant of BIV or whether it was acquired by exposure to amphibians co-inhabiting the same facility and originating from different geographic locations. Lastly, several surviving toads remained PCR-positive 10 wk after the conclusion of the outbreak. This finding has implications for the management of amphibians destined for use in reintroduction programs, as their release may inadvertently lead to viral dissemination.

Differential transcription of fathead minnow immune-related genes following infection with frog virus 3, an emerging pathogen of ectothermic vertebrates.

Frog virus 3 (FV3) and other ranaviruses are responsible for die-offs involving wild, farmed, and captive amphibians, fish, and reptiles. To ascertain which elements of the immune system respond to infection, we explored transcriptional responses following infection of fathead minnow cells with either wild type (wt) FV3 or a knock out (KO) mutant targeting the 18 kDa immediate early gene (18K). At 8h post infection we observed marked upregulation of multiple transcripts encoding proteins affecting innate and acquired immunity. Sequences expressed 4-fold or higher in wt-infected cells included transcripts encoding interferon (IFN), IFN regulatory factors (IRFs), IFN stimulated genes (ISGs) such as Mx and MHC class I, and interleukins IL-1ß, IL-8, IL-17C and IL-12. Cells infected with the 18K KO mutant (∆18K) showed qualitative differences and lower levels of induction. Collectively, these results indicate that ranavirus infection induced expression of multiple cellular genes affecting both innate and acquired immunity.

The molecular biology of frog virus 3 and other iridoviruses infecting cold-blooded vertebrates.

Frog virus 3 (FV3) is the best characterized member of the family Iridoviridae. FV3 study has provided insights into the replication of other family members, and has served as a model of viral transcription, genome replication, and virus-mediated host-shutoff. Although the broad outlines of FV3 replication have been elucidated, the precise roles of most viral proteins remain unknown. Current studies using knock down (KD) mediated by antisense morpholino oligonucleotides (asMO) and small, interfering RNAs (siRNA), knock out (KO) following replacement of the targeted gene with a selectable marker by homologous recombination, ectopic viral gene expression, and recombinant viral proteins have enabled researchers to systematically ascertain replicative- and virulence-related gene functions. In addition, the application of molecular tools to ecological studies is providing novel ways for field biologists to identify potential pathogens, quantify infections, and trace the evolution of ecologically important viral species. In this review, we summarize current studies using not only FV3, but also other iridoviruses infecting ectotherms. As described below, general principles ascertained using FV3 served as a model for the family, and studies utilizing other ranaviruses and megalocytiviruses have confirmed and extended our understanding of iridovirus replication. Collectively, these and future efforts will elucidate molecular events in viral replication, intrinsic and extrinsic factors that contribute to disease outbreaks, and the role of the host immune system in protection from disease.

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.

Characterization of a ranavirus inhibitor of the antiviral protein kinase PKR.

BACKGROUND: Ranaviruses (family Iridoviridae) are important pathogens of lower vertebrates. However, little is known about how they circumvent the immune response of their hosts. Many ranaviruses contain a predicted protein, designated vIF2α, which shows homology with the eukaryotic translation initiation factor 2α. In analogy to distantly related proteins found in poxviruses vIF2α might act as an inhibitor of the antiviral protein kinase PKR. RESULTS: We have characterized the function of vIF2α from Rana catesbeiana virus Z (RCV-Z). Multiple sequence alignments and secondary structure prediction revealed homology of vIF2α with eIF2α throughout the S1-, helical- and C-terminal domains. Genetic and biochemical analyses showed that vIF2α blocked the toxic effects of human and zebrafish PKR in a heterologous yeast system. Rather than complementing eIF2α function, vIF2α acted in a manner comparable to the vaccinia virus (VACV) K3L protein (K3), a pseudosubstrate inhibitor of PKR. Both vIF2α and K3 inhibited human PKR-mediated eIF2α phosphorylation, but not PKR autophosphorylation on Thr446. In contrast the E3L protein (E3), another poxvirus inhibitor of PKR, inhibited both Thr446 and eIF2α Ser51 phosphorylation. Interestingly, phosphorylation of eIF2α by zebrafish PKR was inhibited by vIF2α and E3, but not by K3. Effective inhibition of PKR activity coincided with increased PKR expression levels, indicative of relieved autoinhibition of PKR expression. Experiments with vIF2α deletion constructs, showed that both the N-terminal and helical domains were sufficient for inhibition of PKR, whereas the C-terminal domain was dispensable. CONCLUSIONS: Our results show that RCV-Z vIF2α is a functional inhibitor of human and zebrafish PKR, and probably functions in similar fashion as VACV K3. This constitutes an important step in understanding the interaction of ranaviruses and the host innate immune system.

Frog virus 3 ORF 53R, a putative myristoylated membrane protein, is essential for virus replication in vitro.

Although previous work identified 12 complementation groups with possible roles in virus assembly, currently only one frog virus 3 protein, the major capsid protein (MCP), has been linked with virion formation. To identify other proteins required for assembly, we used an antisense morpholino oligonucleotide to target 53R, a putative myristoylated membrane protein, and showed that treatment resulted in marked reductions in 53R levels and a 60% drop in virus titers. Immunofluorescence assays confirmed knock down and showed that 53R was found primarily within viral assembly sites, whereas transmission electron microscopy detected fewer mature virions and, in some cells, dense granular bodies that may represent unencapsidated DNA-protein complexes. Treatment with a myristoylation inhibitor (2-hydroxymyristic acid) resulted in an 80% reduction in viral titers. Collectively, these data indicate that 53R is an essential viral protein that is required for replication in vitro and suggest it plays a critical role in virion formation.

Identification and expression analyses of poly [I:C]-stimulated genes in channel catfish (Ictalurus punctatus).

Channel catfish (Ictalurus punctatus) have proven to be an excellent model with which to study immune responses of lower vertebrates. Identification of anti-viral antibodies and cytotoxic cells, as well as both type I and II interferon (IFN), demonstrates that catfish likely mount a vigorous anti-viral immune response. In this report, we focus on other elements of the anti-viral response, and identify more than two dozen genes that are induced following treatment of catfish cells with poly [I:C]. We showed that poly [I:C] induced type I interferon within 2 h of treatment, and that characteristic interferon stimulated genes (ISGs) appeared 6-12 h after exposure. Among the ISGs detected by RT-PCR assay were homologs of ISG15, Mx1, IFN regulatory factor 1 (IRF-1), inhibitor of apoptosis protein-1 (IAP-1) and the chemokine CXCL10. Microarray analyses showed that 13 and 24 cellular genes, respectively, were upregulated in poly [I:C]-treated B cell and fibroblast cultures. Although many of these genes were novel and did not fit the profile of mammalian ISGs, there were several (ISG-15, ubiquitin-conjugating enzyme E2G1, integrin-linked kinase, and clathrin-associated protein 47) that were identified as ISGs in mammalian systems. Taken together, these results suggest that dsRNA, either directly or through the prior induction of IFN, upregulates catfish gene products that function individually and/or collectively to inhibit virus replication.

Inhibition of iridovirus protein synthesis and virus replication by antisense morpholino oligonucleotides targeted to the major capsid protein, the 18 kDa immediate-early protein, and a viral homolog of RNA polymerase II.

Frog virus 3 (FV3) is a large DNA virus that encodes approximately 100 proteins. Although the general features of FV3 replication are known, the specific roles that most viral proteins play in the virus life cycle have not yet been elucidated. To address the question of viral gene function, antisense morpholino oligonucleotides (asMOs) were used to transiently knock-down expression of specific viral genes and thus infer their role in virus replication. We designed asMOs directed against the major capsid protein (MCP), an 18 kDa immediate-early protein (18K) that was thought to be a viral regulatory protein, and the viral homologue of the largest subunit of RNA polymerase II (vPol-IIalpha). All three asMOs successfully inhibited translation of the targeted protein, and two of the three asMOs resulted in marked phenotypic changes. Knock-down of the MCP resulted in a marked reduction in viral titer without a corresponding drop in the synthesis of other late viral proteins. Transmission electron microscopy (TEM) showed that in cells treated with the anti-MCP MO assembly sites were devoid of viral particles and contained numerous aberrant structures. In contrast, inhibition of 18K synthesis did not block virion formation, suggesting that the 18K protein was not essential for replication of FV3 in fathead minnow (FHM) cells. Finally, consistent with the view that late viral gene expression is catalyzed by a virus-encoded or virus-modified Pol-II-like protein, knock-down of vPol-IIalpha triggered a global decline in late gene expression and virus yields without affecting the synthesis of early viral genes. Collectively, these results demonstrate the utility of using asMOs to elucidate the function of FV3 proteins.

Rana catesbeiana virus Z (RCV-Z): a novel pathogenic ranavirus.

A virus, designated Rana catesbeiana virus Z (RCV-Z), was isolated from the visceral tissue of moribund tadpoles of the North American bullfrog Rana catesbeiana. SDS-PAGE (sodium dodecyl sulfate polyacrylamide gel electrophoresis) analysis of viral proteins and sequence analysis of the amino terminal end of the major capsid protein showed that RCV-Z was similar to frog virus 3 (FV3) and other ranaviruses isolated from anurans and fish. However, analysis of restriction fragment profiles following digestion of viral genomic DNA with XbaI and BamHI indicated that RCV-Z was markedly different from FV3. Moreover, in contrast to FV3, RCV-Z contained a full-length copy of the viral homolog of eukaryotic initiation factor 2 alpha (eIF-2alpha). Experimental infection of bullfrog tadpoles with FV3 and RCV-Z demonstrated that RCV-Z was much more pathogenic than FV3, and that prior infection with FV3 protected them from subsequent RCV-Z induced mortality. Collectively, these results suggest that RCV-Z may represent a novel species of ranavirus capable of infecting frogs and that possession of a viral eIF-2alpha homolog (vIF-2alpha) correlates with enhanced virulence.