Special Issue: Viruses Infecting Fish, Amphibians, and Reptiles

Although viruses infecting and affecting humans are the focus of considerable research effort, viruses that target other animal species, including cold-blooded vertebrates, are receiving increased attention. In part this reflects the interests of comparative virologists, but increasingly it is based on the impact that many viruses have on ecologically and commercially important animals. Frogs and other amphibians are sentinels of environmental health and their disappearance following viral or fungal (chytrid) infection is a cause for alarm. Likewise, because aquaculture and mariculture are providing an increasingly large percentage of the “seafood” consumed by humans, viral agents that adversely impact the harvest of cultured fish and amphibians are of equal concern. [...

Introduction: History and Future of Ranaviruses

Dr. Allan Granoff (1923–2012), who isolated the first ranavirus (Granoff et al. 1966), had, scattered throughout his office at St. Jude Children’s Research Hospital, a variety of frog-related items including the poem cited above. Although one of Allan’s isolates, Frog virus 3 (FV3), subsequently became the best-characterized member of both the genus (Ranavirus) and the family (Iridoviridae); the impact of that discovery was not fully appreciated at the time. FV3 was neither the first iridoviridae to be recognized as a pathogen of lower vertebrates or the first isolated. Those honors belonged to lymphocystis disease virus (LCDV) and Invertebrate iridovirus 1 (IIV1), respectively (Wissenberg 1965; Xeros 1954). LCDV is responsible for a generally non-life threatening, but disfiguring, disease in fish characterized by the appearance of wart-like growths on the skin and (rarely) internal organs, whereas IIV1 is the causative agent of latent and patent infections in crane fly larvae. Despite its lack of primacy, FV3 was studied because, in keeping with the mission of St. Jude Hospital, it was initially thought to be linked to adenocarcinoma in frogs and thus could be a useful model of human malignancies. Furthermore, unlike LCDV and IIV1, it could be readily grown in cultured cells and was thus amenable to detailed molecular characterization. Although its role in tumor development was soon proven incorrect, FV3 served as a gateway into understanding the replication strategy of a heretofore poorly studied virus family. Moreover, over the next 20 years, its study led to important insights not only into iridoviridae replication, but also eukaryotic biology, virus evolution, and host–virus interactions

THIRD INTERNATIONAL SYMPOSIUM ON RANAVIRUSES:: ADVANCING THE UNDERSTANDING OF THE THREAT OF RANAVIRUSES TO NORTH AMERICAN HERPETOFAUNA

Members of the genus Ranavirus, one of five genera withinthe family Iridoviridae, encompass a group of large, doublestrandedDNA viruses that infect all three classes of ectothermicvertebrates: fish, amphibians, and reptiles. Ranaviruses areglobally emerging pathogens that cause considerable morbidityand mortality among diverse populations. In North America,ranavirus epizootics are regularly reported in wild and culturedfish, amphibian, and reptile populations

Characterization of a ranavirus inhibitor of the antiviral protein kinase PKR

<p>Abstract</p> <p>Background</p> <p>Ranaviruses (family <it>Iridoviridae</it>) 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.</p> <p>Results</p> <p>We have characterized the function of vIF2α from <it>Rana catesbeiana </it>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.</p> <p>Conclusions</p> <p>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.</p

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

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.</p

Identification of a Novel Marine Fish Virus, Singapore Grouper Iridovirus-Encoded MicroRNAs Expressed in Grouper Cells by Solexa Sequencing

BACKGROUND: MicroRNAs (miRNAs) are ubiquitous non-coding RNAs that regulate gene expression at the post-transcriptional level. An increasing number of studies has revealed that viruses can also encode miRNAs, which are proposed to be involved in viral replication and persistence, cell-mediated antiviral immune response, angiogenesis, and cell cycle regulation. Singapore grouper iridovirus (SGIV) is a pathogenic iridovirus that has severely affected grouper aquaculture in China and Southeast Asia. Comprehensive knowledge about the related miRNAs during SGIV infection is helpful for understanding the infection and the pathogenic mechanisms. METHODOLOGY/PRINCIPAL FINDINGS: To determine whether SGIV encoded miRNAs during infection, a small RNA library derived from SGIV-infected grouper (GP) cells was constructed and sequenced by Illumina/Solexa deep-sequencing technology. We recovered 6,802,977 usable reads, of which 34,400 represented small RNA sequences encoded by SGIV. Sixteen novel SGIV-encoded miRNAs were identified by a computational pipeline, including a miRNA that shared a similar sequence to herpesvirus miRNA HSV2-miR-H4-5p, which suggests miRNAs are conserved in far related viruses. Generally, these 16 miRNAs are dispersed throughout the SGIV genome, whereas three are located within the ORF057L region. Some SGIV-encoded miRNAs showed marked sequence and length heterogeneity at their 3' and/or 5' end that could modulate their functions. Expression levels and potential biological activities of these viral miRNAs were examined by stem-loop quantitative RT-PCR and luciferase reporter assay, respectively, and 11 of these viral miRNAs were present and functional in SGIV-infected GP cells. CONCLUSIONS: Our study provided a genome-wide view of miRNA production for iridoviruses and identified 16 novel viral miRNAs. To the best of our knowledge, this is the first experimental demonstration of miRNAs encoded by aquatic animal viruses. The results provide a useful resource for further in-depth studies on SGIV infection and iridovirus pathogenesis

Double-stranded RNA-activated protein kinase PKR of fishes and amphibians: Varying the number of double-stranded RNA binding domains and lineage-specific duplications

BackgroundDouble-stranded (ds) RNA, generated during viral infection, binds and activates the mammalian anti-viral protein kinase PKR, which phosphorylates the translation initiation factor eIF2alpha leading to the general inhibition of protein synthesis. Although PKR-like activity has been described in fish cells, the responsible enzymes eluded molecular characterization until the recent discovery of goldfish and zebrafish PKZ, which contain Z-DNA-binding domains instead of dsRNA-binding domains (dsRBDs). Fish and amphibian PKR genes have not been described so far.ResultsHere we report the cloning and identification of 13 PKR genes from 8 teleost fish and amphibian species, including zebrafish, demonstrating the coexistence of PKR and PKZ in this latter species. Analyses of their genomic organization revealed up to three tandemly arrayed PKR genes, which are arranged in head-to-tail orientation. At least five duplications occurred independently in fish and amphibian lineages. Phylogenetic analyses reveal that the kinase domains of fish PKR genes are more closely related to those of fish PKZ than to the PKR kinase domains of other vertebrate species. The duplication leading to fish PKR and PKZ genes occurred early during teleost fish evolution after the divergence of the tetrapod lineage. While two dsRBDs are found in mammalian and amphibian PKR, one, two or three dsRBDs are present in fish PKR. In zebrafish, both PKR and PKZ were strongly upregulated after immunostimulation with some tissue-specific expression differences. Using genetic and biochemical assays we demonstrate that both zebrafish PKR and PKZ can phosphorylate eIF2alpha in yeast.ConclusionConsidering the important role for PKR in host defense against viruses, the independent duplication and fixation of PKR genes in different lineages probably provided selective advantages by leading to the recognition of an extended spectrum of viral nucleic acid structures, including both dsRNA and Z-DNA/RNA, and perhaps by altering sensitivity to viral PKR inhibitors. Further implications of our findings for the evolution of the PKR family and for studying PKR/PKZ interactions with viral gene products and their roles in viral infections are discussed

The impact of co-infections on fish: a review

International audienceAbstractCo-infections are very common in nature and occur when hosts are infected by two or more different pathogens either by simultaneous or secondary infections so that two or more infectious agents are active together in the same host. Co-infections have a fundamental effect and can alter the course and the severity of different fish diseases. However, co-infection effect has still received limited scrutiny in aquatic animals like fish and available data on this subject is still scarce. The susceptibility of fish to different pathogens could be changed during mixed infections causing the appearance of sudden fish outbreaks. In this review, we focus on the synergistic and antagonistic interactions occurring during co-infections by homologous or heterologous pathogens. We present a concise summary about the present knowledge regarding co-infections in fish. More research is needed to better understand the immune response of fish during mixed infections as these could have an important impact on the development of new strategies for disease control programs and vaccination in fish