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Editorial

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3 pages, 171 KiB

Open AccessEditorial

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

by V. Gregory Chinchar and Amanda L. J. Duffus

Viruses 2019, 11(6), 538; https://doi.org/10.3390/v11060538 - 9 Jun 2019

Cited by 10 | Viewed by 2407

Abstract

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 [...] Full article

(This article belongs to the Special Issue Family Iridoviridae: Molecular and Ecological Studies of a Family Infecting Invertebrates and Ectothermic Vertebrates)

Research

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25 pages, 3349 KiB

Open AccessArticle

Detection and Characterization of Invertebrate Iridoviruses Found in Reptiles and Prey Insects in Europe over the Past Two Decades

by Tibor Papp and Rachel E. Marschang

Viruses 2019, 11(7), 600; https://doi.org/10.3390/v11070600 - 2 Jul 2019

Cited by 23 | Viewed by 4030

Abstract

Invertebrate iridoviruses (IIVs), while mostly described in a wide range of invertebrate hosts, have also been repeatedly detected in diagnostic samples from poikilothermic vertebrates including reptiles and amphibians. Since iridoviruses from invertebrate and vertebrate hosts differ strongly from one another based not only [...] Read more.

Invertebrate iridoviruses (IIVs), while mostly described in a wide range of invertebrate hosts, have also been repeatedly detected in diagnostic samples from poikilothermic vertebrates including reptiles and amphibians. Since iridoviruses from invertebrate and vertebrate hosts differ strongly from one another based not only on host range but also on molecular characteristics, a series of molecular studies and bioassays were performed to characterize and compare IIVs from various hosts and evaluate their ability to infect a vertebrate host. Eight IIV isolates from reptilian and orthopteran hosts collected over a period of six years were partially sequenced. Comparison of eight genome portions (total over 14 kbp) showed that these were all very similar to one another and to an earlier described cricket IIV isolate, thus they were given the collective name lizard–cricket IV (Liz–CrIV). One isolate from a chameleon was also subjected to Illumina sequencing and almost the entire genomic sequence was obtained. Comparison of this longer genome sequence showed several differences to the most closely related IIV, Invertebrate iridovirus 6 (IIV6), the type species of the genus Iridovirus, including several deletions and possible recombination sites, as well as insertions of genes of non-iridoviral origin. Three isolates from vertebrate and invertebrate hosts were also used for comparative studies on pathogenicity in crickets (Gryllus bimaculatus) at 20 and 30 °C. Finally, the chameleon isolate used for the genome sequencing studies was also used in a transmission study with bearded dragons. The transmission studies showed large variability in virus replication and pathogenicity of the three tested viruses in crickets at the two temperatures. In the infection study with bearded dragons, lizards inoculated with a Liz–CrIV did not become ill, but the virus was detected in numerous tissues by qPCR and was also isolated in cell culture from several tissues. Highest viral loads were measured in the gastro-intestinal organs and in the skin. These studies demonstrate that Liz–CrIV circulates in the pet trade in Europe. This virus is capable of infecting both invertebrates and poikilothermic vertebrates, although its involvement in disease in the latter has not been proven. Full article

(This article belongs to the Special Issue Family Iridoviridae: Molecular and Ecological Studies of a Family Infecting Invertebrates and Ectothermic Vertebrates)

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11 pages, 1956 KiB

Open AccessArticle

Ranaviruses Bind Cells from Different Species through Interaction with Heparan Sulfate

by Fei Ke, Zi-Hao Wang, Cheng-Yue Ming and Qi-Ya Zhang

Viruses 2019, 11(7), 593; https://doi.org/10.3390/v11070593 - 29 Jun 2019

Cited by 14 | Viewed by 2721

Abstract

Ranavirus cross-species infections have been documented, but the viral proteins involved in the interaction with cell receptors have not yet been identified. Here, viral cell-binding proteins and their cognate cellular receptors were investigated using two ranaviruses, Andrias davidianus ranavirus (ADRV) and Rana grylio [...] Read more.

Ranavirus cross-species infections have been documented, but the viral proteins involved in the interaction with cell receptors have not yet been identified. Here, viral cell-binding proteins and their cognate cellular receptors were investigated using two ranaviruses, Andrias davidianus ranavirus (ADRV) and Rana grylio virus (RGV), and two different cell lines, Chinese giant salamander thymus cells (GSTC) and Epithelioma papulosum cyprinid (EPC) cells. The heparan sulfate (HS) analog heparin inhibited plaque formation of ADRV and RGV in the two cell lines by more than 80% at a concentration of 5 μg/mL. In addition, enzymatic removal of cell surface HS by heparinase I markedly reduced plaque formation by both viruses and competition with heparin reduced virus-cell binding. These results indicate that cell surface HS is involved in ADRV and RGV cell binding and infection. Furthermore, recombinant viral envelope proteins ADRV-58L and RGV-53R bound heparin-Sepharose beads implying the potential that cell surface HS is involved in the initial interaction between ranaviruses and susceptible host cells. To our knowledge, this is the first report identifying cell surface HS as ranavirus binding factor and furthers understanding of interactions between ranaviruses and host cells. Full article

(This article belongs to the Special Issue Family Iridoviridae: Molecular and Ecological Studies of a Family Infecting Invertebrates and Ectothermic Vertebrates)

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Soluble heparin inhibits Andrias davidianus ranavirus (ADRV) and Rana grylio virus (RGV) infection of giant salamander thymus cells (GSTC) and Epithelioma papulosum cyprinid (EPC) cells. Cells were infected with ADRV or RGV that had been pre-incubated in the presence of different concentrations of heparin. The number of plaques obtained in the absence of heparin was set as 1. The data represent triplicate results and was analyzed with Student’s t-test. Significant differences (versus virus without heparin) are marked with * (p < 0.05). Full article ">Figure 2
Heparan sulfate (a) and chondroitin sulfate (b) inhibit ADRV and RGV infection of GSTC and EPC cells. GSTC and EPC cells were infected with ADRV or RGV in the presence of different concentrations of heparan sulfate and chondroitin sulfate. The number of plaques obtained without glycosaminoglycans (GAGs) was set as 1. Experiments were conducted in triplicate and analyzed using Student’s t-test. Significant differences (versus virus without exposure to GAGs) are marked with * (p < 0.05). Full article ">Figure 3
Heparinase I treatment reduced ADRV and RGV plaque formation in GSTC cells. Cells were infected with ADRV or RGV after treatment with different concentrations of heparinase I and plaque formation monitored. Plaque numbers obtained in the absence of heparinase treatment were set as 1. Triplicate results were analyzed by Student’s t-test, and significant differences are marked with * (p < 0.05). Full article ">Figure 4
Soluble heparin inhibits virus binding to GSTC cells. Viral suspensions or purified virions were added to GSTC cells in the presence of different concentrations of heparin. After incubation, virion binding was assessed by determining the number of bound viral genomes by qPCR. DNA levels observed in the absence of heparin pre-treatment were set as 1. The data were obtained from three experiments and analyzed with Student’s t-test. Significant differences are marked with * (p < 0.05). Full article ">Figure 5
Recombinant proteins bind heparin-Sepharose beads. (a) Schematic diagram of the recombinant proteins: The N-terminal domain of ADRV-58L (r58L-N), the N-terminal domain of RGV-53R (r53R-N), and the C-terminal domain of ADRV-58L (r58L-C) were expressed using pET32a or pET28a. The predicted transmembrane region is shown in the grey box. (b) Expression and purification of the three proteins (r58L-N, r53R-N, and r58L-C) with pET32a vector. M: protein marker; 1, 4, 8: Bacteria without induction; 2, 5, 9: Bacteria with induction; 3, 6, 10: Purified proteins. The recombinant proteins are indicated with asterisks, and their predicted molecular weights are shown on the right. (c) Expression and purification of the three proteins using pET28a. M: Protein marker; 1, 5, 10: Cacteria without induction; 2, 6, 11: Cacteria with induction; 3, 4, 7, 8, 12: Purified proteins. The recombinant proteins are indicated with asterisks, and their predicted molecular weights are shown on the right. (d) Binding of recombinant proteins and heparin-Sepharose beads. Recombinant proteins were incubated with heparin-Sepharose or Sepharose beads. The fractions of input (Input), supernatant after incubation (S), the fifth wash solution (W5), and the eluate (Eluate) were detected by Western blot with the anti-His antibody. Recombinant proteins expressed with pET32a or pET28a vectors were used. Recombinant proteins were observed in the Input and Elute fractions from heparin-Sepharose beads and S fraction from control beads. Full article ">

13 pages, 2481 KiB

Open AccessArticle

Modelling Ranavirus Transmission in Populations of Common Frogs (Rana temporaria) in the United Kingdom

by Amanda L.J. Duffus, Trenton W.J. Garner, Richard A. Nichols, Joshua P. Standridge and Julia E. Earl

Viruses 2019, 11(6), 556; https://doi.org/10.3390/v11060556 - 15 Jun 2019

Cited by 6 | Viewed by 3870

Abstract

Ranaviruses began emerging in common frogs (Rana temporaria) in the United Kingdom in the late 1980s and early 1990s, causing severe disease and declines in the populations of these animals. Herein, we explored the transmission dynamics of the ranavirus(es) present in [...] Read more.

Ranaviruses began emerging in common frogs (Rana temporaria) in the United Kingdom in the late 1980s and early 1990s, causing severe disease and declines in the populations of these animals. Herein, we explored the transmission dynamics of the ranavirus(es) present in common frog populations, in the context of a simple susceptible-infected (SI) model, using parameters derived from the literature. We explored the effects of disease-induced population decline on the dynamics of the ranavirus. We then extended the model to consider the infection dynamics in populations exposed to both ulcerative and hemorrhagic forms of the ranaviral disease. The preliminary investigation indicated the important interactions between the forms. When the ulcerative form was present in a population and the hemorrhagic form was later introduced, the hemorrhagic form of the disease needed to be highly contagious, to persist. We highlighted the areas where further research and experimental evidence is needed and hope that these models would act as a guide for further research into the amphibian disease dynamics. Full article

(This article belongs to the Special Issue Family Iridoviridae: Molecular and Ecological Studies of a Family Infecting Invertebrates and Ectothermic Vertebrates)

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10 pages, 783 KiB

Open AccessArticle

Artemia spp., a Susceptible Host and Vector for Lymphocystis Disease Virus

by Estefania J. Valverde, Alejandro M. Labella, Juan J. Borrego and Dolores Castro

Viruses 2019, 11(6), 506; https://doi.org/10.3390/v11060506 - 1 Jun 2019

Cited by 11 | Viewed by 3035

Abstract

Different developmental stages of Artemia spp. (metanauplii, juveniles and adults) were bath-challenged with two isolates of the Lymphocystis disease virus (LCDV), namely, LCDV SA25 (belonging to the species Lymphocystis disease virus 3) and ATCC VR-342 (an unclassified member of the genus Lymphocystivirus [...] Read more.

Different developmental stages of Artemia spp. (metanauplii, juveniles and adults) were bath-challenged with two isolates of the Lymphocystis disease virus (LCDV), namely, LCDV SA25 (belonging to the species Lymphocystis disease virus 3) and ATCC VR-342 (an unclassified member of the genus Lymphocystivirus). Viral quantification and gene expression were analyzed by qPCR at different times post-inoculation (pi). In addition, infectious titres were determined at 8 dpi by integrated cell culture (ICC)-RT-PCR, an assay that detects viral mRNA in inoculated cell cultures. In LCDV-challenged Artemia, the viral load increased by 2–3 orders of magnitude (depending on developmental stage and viral isolate) during the first 8–12 dpi, with viral titres up to 2.3 × 10² Most Probable Number of Infectious Units (MPNIU)/mg. Viral transcripts were detected in the infected Artemia, relative expression values showed a similar temporal evolution in the different experimental groups. Moreover, gilthead seabream (Sparus aurata) fingerlings were challenged by feeding on LCDV-infected metanauplii. Although no Lymphocystis symptoms were observed in the fish, the number of viral DNA copies was significantly higher at the end of the experimental trial and major capsid protein (mcp) gene expression was consistently detected. The results obtained support that LCDV infects Artemia spp., establishing an asymptomatic productive infection at least under the experimental conditions tested, and that the infected metanauplii are a vector for LCDV transmission to gilthead seabream. Full article

(This article belongs to the Special Issue Family Iridoviridae: Molecular and Ecological Studies of a Family Infecting Invertebrates and Ectothermic Vertebrates)

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10 pages, 2382 KiB

Open AccessArticle

Characterization of a Novel Megalocytivirus Isolated from European Chub (Squalius cephalus)

by Maya A. Halaly, Kuttichantran Subramaniam, Samantha A. Koda, Vsevolod L. Popov, David Stone, Keith Way and Thomas B. Waltzek

Viruses 2019, 11(5), 440; https://doi.org/10.3390/v11050440 - 15 May 2019

Cited by 14 | Viewed by 4366

Abstract

A novel virus from moribund European chub (Squalius cephalus) was isolated on epithelioma papulosum cyprini (EPC) cells. Transmission electron microscopic examination revealed abundant non-enveloped, hexagonal virus particles in the cytoplasm of infected EPC cells consistent with an iridovirus. Illumina MiSeq sequence [...] Read more.

A novel virus from moribund European chub (Squalius cephalus) was isolated on epithelioma papulosum cyprini (EPC) cells. Transmission electron microscopic examination revealed abundant non-enveloped, hexagonal virus particles in the cytoplasm of infected EPC cells consistent with an iridovirus. Illumina MiSeq sequence data enabled the assembly and annotation of the full genome (128,216 bp encoding 108 open reading frames) of the suspected iridovirus. Maximum Likelihood phylogenetic analyses based on 25 iridovirus core genes supported the European chub iridovirus (ECIV) as being the sister species to the recently-discovered scale drop disease virus (SDDV), which together form the most basal megalocytivirus clade. Genetic analyses of the ECIV major capsid protein and ATPase genes revealed the greatest nucleotide identity to members of the genus Megalocytivirus including SDDV. These data support ECIV as a novel member within the genus Megalocytivirus. Experimental challenge studies are needed to fulfill River’s postulates and determine whether ECIV induces the pathognomonic microscopic lesions (i.e., megalocytes with basophilic cytoplasmic inclusions) observed in megalocytivirus infections. Full article

(This article belongs to the Special Issue Family Iridoviridae: Molecular and Ecological Studies of a Family Infecting Invertebrates and Ectothermic Vertebrates)

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13 pages, 4459 KiB

Open AccessArticle

Interaction between Two Iridovirus Core Proteins and Their Effects on Ranavirus (RGV) Replication in Cells from Different Species

by Xiao-Tao Zeng and Qi-Ya Zhang

Viruses 2019, 11(5), 416; https://doi.org/10.3390/v11050416 - 4 May 2019

Cited by 12 | Viewed by 3313

Abstract

The two putative proteins RGV-63R and RGV-91R encoded by Rana grylio virus (RGV) are DNA polymerase and proliferating cell nuclear antigen (PCNA) respectively, and are core proteins of iridoviruses. Here, the interaction between RGV-63R and RGV-91R was detected by a yeast two-hybrid (Y2H) [...] Read more.

The two putative proteins RGV-63R and RGV-91R encoded by Rana grylio virus (RGV) are DNA polymerase and proliferating cell nuclear antigen (PCNA) respectively, and are core proteins of iridoviruses. Here, the interaction between RGV-63R and RGV-91R was detected by a yeast two-hybrid (Y2H) assay and further confirmed by co-immunoprecipitation (co-IP) assays. Subsequently, RGV-63R or RGV-91R were expressed alone or co-expressed in two kinds of aquatic animal cells including amphibian Chinese giant salamander thymus cells (GSTCs) and fish Epithelioma papulosum cyprinid cells (EPCs) to investigate their localizations and effects on RGV genome replication. The results showed that their localizations in the two kinds of cells are consistent. RGV-63R localized in the cytoplasm, while RGV-91R localized in the nucleus. However, when co-expressed, RGV-63R localized in both the cytoplasm and the nucleus, and colocalized with RGV-91R in the nucleus. 91R△NLS represents the RGV-91R deleting nuclear localization signal, which is localized in the cytoplasm and colocalized with RGV-63R in the cytoplasm. qPCR analysis revealed that sole expression and co-expression of the two proteins in the cells of two species significantly promoted RGV genome replication, while varying degrees of viral genome replication levels may be linked to the cell types. This study provides novel molecular evidence for ranavirus cross-species infection and replication. Full article

(This article belongs to the Special Issue Family Iridoviridae: Molecular and Ecological Studies of a Family Infecting Invertebrates and Ectothermic Vertebrates)

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15 pages, 2213 KiB

Open AccessArticle

IIV-6 Inhibits NF-κB Responses in Drosophila

by Cara West, Florentina Rus, Ying Chen, Anni Kleino, Monique Gangloff, Don B. Gammon and Neal Silverman

Viruses 2019, 11(5), 409; https://doi.org/10.3390/v11050409 - 1 May 2019

Cited by 10 | Viewed by 5101

Abstract

The host immune response and virus-encoded immune evasion proteins pose constant, mutual selective pressure on each other. Virally encoded immune evasion proteins also indicate which host pathways must be inhibited to allow for viral replication. Here, we show that IIV-6 is capable of [...] Read more.

The host immune response and virus-encoded immune evasion proteins pose constant, mutual selective pressure on each other. Virally encoded immune evasion proteins also indicate which host pathways must be inhibited to allow for viral replication. Here, we show that IIV-6 is capable of inhibiting the two Drosophila NF-κB signaling pathways, Imd and Toll. Antimicrobial peptide (AMP) gene induction downstream of either pathway is suppressed when cells infected with IIV-6 are also stimulated with Toll or Imd ligands. We find that cleavage of both Imd and Relish, as well as Relish nuclear translocation, three key points in Imd signal transduction, occur in IIV-6 infected cells, indicating that the mechanism of viral inhibition is farther downstream, at the level of Relish promoter binding or transcriptional activation. Additionally, flies co-infected with both IIV-6 and the Gram-negative bacterium, Erwinia carotovora carotovora, succumb to infection more rapidly than flies singly infected with either the virus or the bacterium. These findings demonstrate how pre-existing infections can have a dramatic and negative effect on secondary infections, and establish a Drosophila model to study confection susceptibility. Full article

(This article belongs to the Special Issue Family Iridoviridae: Molecular and Ecological Studies of a Family Infecting Invertebrates and Ectothermic Vertebrates)

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IIV-6 inhibits Imd and Toll Signaling. (A) S2* cells were treated with 20-hydroxyecdysone (EcD) as indicated for 18 hours and then infected with IIV-6 (blue circles) or uninfected (black circles) for six hours. Cells were then stimulated with DAP-type PGN for six hours, where indicated. Diptericin (Dpt) levels were monitored by qRT-PCR. (B) S2* cells were treated with 20-hydroxyecdysone (EcD) as indicated for 18 h and then infected with IIV-6 (blue circles) or uninfected (black circles) for six hours. Cells were then stimulated with cleaved Spätzle (spz) for 18 h, where indicated. Drosomycin (Drs) levels were monitored by qRT-PCR. (A,B) Black bars indicate mean and error bars indicate standard deviation. Statistics were determined using two-way ANOVA and Sidak’s multiple comparisons test; ns, not significant; ****, p < 0.0001. Full article ">Figure 2
Both Imd and Toll regulated AMPs are suppressed by IIV-6 infection. S2* cells were treated with 20-hydroxyecdysone (EcD) for 18 h, and then infected with IIV-6 for 6 h. (A) Cells were stimulated with peptidoglycan (PGN) for 6 h prior to RNA isolation. (B) S2* cells were then stimulated with cleaved Spätzle (spz) for 18 h prior to RNA isolation, and then analyzed by Nanostring nCounter. Heatmaps display Z scores of mRNA levels of immune genes in the presence or absence of virus and pathway stimulation clustered by expression pattern. Biologically independent duplicates are shown. Untr., untreated cells. EcD, cells treated with 20-hydroxyecdysone. EcD + PGN, cells treated with 20-hydroxyecdysone and peptidoglycan. EcD + spz, cells treated with 20-hydroxyecdysone and Spätzle. Full article ">Figure 3
Some AMPs are elevated in vivo upon IIV-6 infection, before returning to baseline. (A) Heatmap of Z score transformed mRNA levels for AMP genes following IIV-6 infection of adult male w1118 flies for the indicated timepoints, assayed by Nanostring nCounter. RNA was isolated from PBS-injected flies at the same time points as a control. Biologically independent samples were analyzed in duplicate. (A’) Detailed comparison of mRNA levels for selected Imd-regulated AMP genes following IIV-6 infection of adult w1118 flies for 12, 24, and 48 h assayed by Nanostring nCounter. Full article ">Figure 4
Imd signaling remains intact in the presence of IIV-6. (A) S2* cells were treated with 20-hydroxyecdysone (EcD) for 18 h. Cells were then infected with IIV-6, where indicated, for six hours. Samples were then stimulated with PGN for 15 min, where indicated, and lysed in standard lysis buffer. Endogenous Imd was monitored by immunoblotting; arrows indicate cleaved (c-Imd) or full-length (FL-Imd) Imd; ns, non-specific. (B) Cells were treated with 20-hydroxyecdysone (EcD), where indicated, for 18 h and infected with IIV-6, as indicated, for six hours. Samples stimulated with EcD were then stimulated with PGN for 15 min, and lysed in standard lysis buffer. Endogenous Relish was probed by immunoblotting using a C-terminal Relish antibody. MOI used was as follows. Lane 2: MOI =2; Lane 4: MOI = 0.2; Lane 5: MOI = 2; Lane 6: MOI = 5. Full article ">Figure 5
Nuclear localization of Relish remains intact in the presence of IIV-6. (A) S2* cells stably expressing YFP-Relish were treated with 20-hydroxyecdysone (EcD) for 2 h and then infected with mCherry-IIV-6 (right) or left uninfected (left) for 18 h. Cells were then stimulated with DAP-type PGN for 15 min (lower panels), prior to fixation and staining with anti-Lamin (shown in green) and Hoechst 33342 (shown in blue). Representative images from four biologically independent experiments are shown. Arrowheads mark cells with nuclear localized Relish. Left panels display the overlay of YFP-Relish, Hoeschst, Lamin, and mCherry-IIV-6 where applicable, while the right panels exhibit the YFP-Relish alone. Scale bars are 10 μm. (B) Quantification of Relish nuclear translocation. Between 400–1800 cells were scored for each condition as displaying Relish localization in the nucleus, in the cytoplasm, or as indeterminate when staining was diffuse throughout both compartments or the signal was too weak to discern. Statistics were determined using two-way ANOVA and Tukey’s multiple comparisons test; *, p < 0.05; ns, not significant. Error bars represent standard deviation. Full article ">Figure 6
Viral replication is not needed for NF-κB inhibition. (A,B) S2* cells were treated with 20-hydroxyecdysone (EcD) as indicated for 18 h and then infected with IIV-6 (blue circles) or uninfected (black circles) for six hours. Cells were then stimulated with DAP-type PGN for six hours, where indicated. Diptericin levels were monitored by qRT-PCR. (A) Cells were treated with cidofovir, a viral polymerase inhibitor, where indicated. Mock cells untreated with EcD, PGN, or cidofovir are shown as a control. Each data point is a biologically independent replicate; n = 3. Error bars represent standard deviation. Statistics were determined using two-way ANOVA and Sidak’s multiple comparisons test; ns, not significant; ****, p < 0.0001. (B) S2* cells were infected with IIV-6 (blue circles), or treated with UV- (purple circles) or heat- (red circles) inactivated IIV-6. Uninfected controls are shown in black. Each data point is a biologically independent replicate; n = 3. Error bars represent standard deviation. Statistics were determined using one-way ANOVA and Tukey’s multiple comparisons test; ns, not significant; ***, p < 0.001. Full article ">Figure 7
Flies infected with IIV-6 have lower AMP levels. (A) Adult w1118 flies were infected with IIV-6, injected with PBS, or left unmanipulated for 7 days, and then snap frozen for RNA isolation. Dpt levels were assayed by qRT-PCR. Black bars indicate mean and error bars indicate standard deviation. Statistics were determined using one-way ANOVA and Tukey’s multiple comparisons test; ns, not significant; *, p = 0.0463; **, p = 0.0033. (B) Kaplan-Meier plots of adult w1118 flies infected with IIV-6 (blue lines) or PBS-injected (black lines) for 8 days prior to bacterial infection. On day 0, flies were pricked with Ecc15 (solid lines) or were sterile pricked (dashed lines) with a microsurgery needle. Flies were counted daily for survivors. Statistics were determined using log-rank test; ****, p < 0.0001. Full article ">

12 pages, 355 KiB

Open AccessArticle

Evaluating the Within-Host Dynamics of Ranavirus Infection with Mechanistic Disease Models and Experimental Data

by Joseph R. Mihaljevic, Amy L. Greer and Jesse L. Brunner

Viruses 2019, 11(5), 396; https://doi.org/10.3390/v11050396 - 27 Apr 2019

Cited by 3 | Viewed by 3294

Abstract

Mechanistic models are critical for our understanding of both within-host dynamics (i.e., pathogen replication and immune system processes) and among-host dynamics (i.e., transmission). Within-host models, however, are not often fit to experimental data, which can serve as a robust method of hypothesis testing [...] Read more.

Mechanistic models are critical for our understanding of both within-host dynamics (i.e., pathogen replication and immune system processes) and among-host dynamics (i.e., transmission). Within-host models, however, are not often fit to experimental data, which can serve as a robust method of hypothesis testing and hypothesis generation. In this study, we use mechanistic models and empirical, time-series data of viral titer to better understand the replication of ranaviruses within their amphibian hosts and the immune dynamics that limit viral replication. Specifically, we fit a suite of potential models to our data, where each model represents a hypothesis about the interactions between viral replication and immune defense. Through formal model comparison, we find a parsimonious model that captures key features of our time-series data: The viral titer rises and falls through time, likely due to an immune system response, and that the initial viral dosage affects both the peak viral titer and the timing of the peak. Importantly, our model makes several predictions, including the existence of long-term viral infections, which can be validated in future studies. Full article

(This article belongs to the Special Issue Family Iridoviridae: Molecular and Ecological Studies of a Family Infecting Invertebrates and Ectothermic Vertebrates)

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15 pages, 3646 KiB

Open AccessArticle

Susceptibility of Exopalaemon carinicauda to the Infection with Shrimp Hemocyte Iridescent Virus (SHIV 20141215), a Strain of Decapod Iridescent Virus 1 (DIV1)

by Xing Chen, Liang Qiu, Hailiang Wang, Peizhuo Zou, Xuan Dong, Fuhua Li and Jie Huang

Viruses 2019, 11(4), 387; https://doi.org/10.3390/v11040387 - 25 Apr 2019

Cited by 53 | Viewed by 6819

Abstract

In this study, ridgetail white prawns—Exopalaemon carinicauda—were infected per os (PO) with debris of Penaeus vannamei infected with shrimp hemocyte iridescent virus (SHIV 20141215), a strain of decapod iridescent virus 1 (DIV1), and via intramuscular injection (IM with raw extracts of [...] Read more.

In this study, ridgetail white prawns—Exopalaemon carinicauda—were infected per os (PO) with debris of Penaeus vannamei infected with shrimp hemocyte iridescent virus (SHIV 20141215), a strain of decapod iridescent virus 1 (DIV1), and via intramuscular injection (IM with raw extracts of SHIV 20141215. The infected E. carinicauda showed obvious clinical symptoms, including weakness, empty gut and stomach, pale hepatopancreas, and partial death with mean cumulative mortalities of 42.5% and 70.8% by nonlinear regression, respectively. Results of TaqMan probe-based real-time quantitative PCR showed that the moribund and surviving individuals with clinical signs of infected E. carinicauda were DIV1-positive. Histological examination showed that there were darkly eosinophilic and cytoplasmic inclusions, of which some were surrounded with or contained tiny basophilic staining, and pyknosis in hemocytes in hepatopancreatic sinus, hematopoietic cells, cuticular epithelium, etc. On the slides of in situ DIG-labeling-loop-mediated DNA amplification (ISDL), positive signals were observed in hematopoietic tissue, stomach, cuticular epithelium, and hepatopancreatic sinus of infected prawns from both PO and IM groups. Transmission electron microscopy (TEM) of ultrathin sections showed that icosahedral DIV1 particles existed in hepatopancreatic sinus and gills of the infected E. carinicauda from the PO group. The viral particles were also observed in hepatopancreatic sinus, gills, pereiopods, muscles, and uropods of the infected E. carinicauda from the IM group. The assembled virions, which mostly distributed along the edge of the cytoplasmic virogenic stromata near cellular membrane of infected cells, were enveloped and approximately 150 nm in diameter. The results of molecular tests, histopathological examination, ISDL, and TEM confirmed that E. carinicauda is a susceptible host of DIV1. This study also indicated that E. carinicauda showed some degree of tolerance to the infection with DIV1 per os challenge mimicking natural pathway. Full article

(This article belongs to the Special Issue Family Iridoviridae: Molecular and Ecological Studies of a Family Infecting Invertebrates and Ectothermic Vertebrates)

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Figure 1
Gross signs of prawns Exopalaemon carinicauda in different groups of the challenge test. CPO: Prawn in per os control group; PO: Prawn in per os group; IM: Prawn in intermuscular injection group. Solid arrows indicate stomach (ST), hepatopancreas (HP), midgut (MG), and hematopoietic tissue (HM). Red bar = 10 mm. Full article ">Figure 2
Cumulative mortalities of Exopalaemon carinicauda in the challenge test. IM group, prawns were challenged with filtrated viral suspension via intermuscular injection; PO group, prawns were fed with tissues of DIV1-infected P. vannamei; CIM group, prawns were injected with sterile PPB-His buffer; CPO group, prawns were fed with commercial feed. Cumulative mortalities are shown as means of data from three replicates for each experimental group (each replicate contained 10 individuals). The mean points with same color indicate no significant difference (P > 0.05), and the mean points with different colors indicate a significant difference (P < 0.05). Overall analysis indicated there are very significant differences among IM, PO, and the two controls (P < 0.01). The curves were drawn based on the nonlinear regression following the three-parameter sigmoid equation. Full article ">Figure 3
Decapod iridescent virus 1 (DIV1) loads in Exopalaemon carinicauda from the challenge test. Different letters above the bars indicate significant difference (P < 0.01). IM: prawn group challenged var intramuscular injection, PO: prawn group challenged per os; CIM: control group of injected with sterile buffer; CPO: control group fed with commercial feed. CIM and CPO were negative. Full article ">Figure 4
Histopathological examination of Exopalaemon carinicauda tissues infected with DIV1 and controls. Black arrows show karyopyknosis and white arrows show eosinophilic inclusions. Pictures (a,b) are hepatopancreas; (c,d) are hematopoietic tissues; and (e,f) are cuticular epithelium on which the cuticles were removed before dehydration. The pictures in the left column (a, c, and e) are the tissues of the infected prawn; the pictures in the right column (b, d, and f) are the tissues of the control prawn. HpT: hepatopancreatic tubule; HpS: hepatopancreatic sinus; Hm: hematopoietic tissue; Ep: epithelium; Cn: connective tissue; m: mitotic phase. Bar = 50 µm. Full article ">Figure 5
Information on primer design and primers used for Loop-mediated isothermal amplification (LAMP), based on the reference sequence of DIV1. Sequences of Shrimp hemocyte iridescent virus (SHIV 20141215 MF599468) and Cherax quadricarinatus iridovirus (CQIV CN01 MF197913) were obtained from GenBank. Full article ">Figure 6
In situ DIG-labeling-loop-mediated DNA amplification (ISDL) micrographs of DIV1-infected and control Exopalaemon carinicauda. Hepatopancreas, stomach, cuticular epithelium, and hematopoietic tissue from a challenged prawn of the PO group, respectively (a, c, e, and g); hepatopancreas, stomach, cuticular epithelium, and hematopoietic tissue from a healthy prawn of the CPO group, respectively (b, d, f, and h). In pictures a, c, e, and g, blue-violet signals were observed in hepatopancreatic sinus, stomach epithelium, cuticular epithelium, and hematopoietic tissues of prawns, respectively. In pictures b, d, f, and h, no hybridization signal was seen in the same tissues of DIV1-negative E. carinicauda, except some non-specific signals on gastric sieve of stomach. Full article ">Figure 7
Transmission electron microscopy (TEM) of DIV1-infected Exopalaemon carinicauda. (A–E): hepatopancreatic sinus, pereiopods, uropods, muscle and gills, respectively, of a prawn in the IM group; (F and G): hepatopancreatic sinus and gills, respectively, of a prawn in the PO group; (H): cytoplasmic inclusions with a cluster of viral particles. Full article ">

16 pages, 3506 KiB

Open AccessArticle

Distribution and Phylogeny of Erythrocytic Necrosis Virus (ENV) in Salmon Suggests Marine Origin

by Veronica A. Pagowski, Gideon J. Mordecai, Kristina M. Miller, Angela D. Schulze, Karia H. Kaukinen, Tobi J. Ming, Shaorong Li, Amy K. Teffer, Amy Tabata and Curtis A. Suttle

Viruses 2019, 11(4), 358; https://doi.org/10.3390/v11040358 - 18 Apr 2019

Cited by 8 | Viewed by 4139

Abstract

Viral erythrocytic necrosis (VEN) affects over 20 species of marine and anadromous fishes in the North Atlantic and North Pacific Oceans. However, the distribution and strain variation of its viral causative agent, erythrocytic necrosis virus (ENV), has not been well characterized within Pacific [...] Read more.

Viral erythrocytic necrosis (VEN) affects over 20 species of marine and anadromous fishes in the North Atlantic and North Pacific Oceans. However, the distribution and strain variation of its viral causative agent, erythrocytic necrosis virus (ENV), has not been well characterized within Pacific salmon. Here, metatranscriptomic sequencing of Chinook salmon revealed that ENV infecting salmon was closely related to ENV from Pacific herring, with inferred amino-acid sequences from Chinook salmon being 99% identical to those reported for herring. Sequence analysis also revealed 89 protein-encoding sequences attributed to ENV, greatly expanding the amount of genetic information available for this virus. High-throughput PCR of over 19,000 fish showed that ENV is widely distributed in the NE Pacific Ocean and was detected in 12 of 16 tested species, including in 27% of herring, 38% of anchovy, 17% of pollock, and 13% of sand lance. Despite frequent detection in marine fish, ENV prevalence was significantly lower in fish from freshwater (0.03%), as assessed with a generalized linear mixed effects model (p = 5.5 × 10⁻⁸). Thus, marine fish are likely a reservoir for the virus. High genetic similarity between ENV obtained from salmon and herring also suggests that transmission between these hosts is likely. Full article

(This article belongs to the Special Issue Family Iridoviridae: Molecular and Ecological Studies of a Family Infecting Invertebrates and Ectothermic Vertebrates)

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Figure 1
Substitution-rate optimized maximum likelihood phylogenetic trees of putative ENV DNA-dependent DNA polymerase (A), DNA-dependent RNA polymerase (B), major capsid protein (C), and ATPase (D) sequences (SEQ#) mapped with related iridoviruses. Alignments were based on nucleotide sequences for the major capsid protein and amino acid sequences for all other trees. Branch labels indicate bootstrap support values for 100 re-samplings and the scale bar indicates substitution rate. GenBank reference sequences and contig IDs are listed below (<a href="#viruses-11-00358-t002" class="html-table">Table 2</a>), with colors indicating genera groups. Putative ENV sequence lengths are listed in <a href="#viruses-11-00358-t001" class="html-table">Table 1</a>. Full article ">Figure 2
Apparent ENV prevalence in mixed tissue smolt samples by species. Error bars indicate 95% confidence intervals. Blue bars indicate proportions with limit of detection (LOD) criteria applied and coral bars indicate proportions without LOD criteria applied. Values indicate sample sizes for each species. Full article ">Figure 3
(A) Full sampling area heat map of the mean calculated ENV copy number, based on interpolated values. Copy number values are binned into 6 numerical categories (see color legend), (B) shows Vancouver Island inset map. Adults and smolts of all species are shown and LOD criteria are not applied. Full article ">Figure 4
Saline (SW) and freshwater (FW) ENV prevalence (A) and load (B) among salmon species and herring. Samples with LOD criteria applied are in blue and samples without LOD criteria applied are in coral. Printed values indicate sample sizes and error bars indicate 95% confidence intervals. Only smolts are shown. Full article ">

14 pages, 3509 KiB

Open AccessArticle

Description of a Natural Infection with Decapod Iridescent Virus 1 in Farmed Giant Freshwater Prawn, Macrobrachium rosenbergii

by Liang Qiu, Xing Chen, Ruo-Heng Zhao, Chen Li, Wen Gao, Qing-Li Zhang and Jie Huang

Viruses 2019, 11(4), 354; https://doi.org/10.3390/v11040354 - 17 Apr 2019

Cited by 72 | Viewed by 8946

Abstract

Macrobrachium rosenbergii is a valuable freshwater prawn in Asian aquaculture. In recent years, a new symptom that was generally called “white head” has caused high mortality in M. rosenbergii farms in China. Samples of M. rosenbergii, M. nipponense, Procambarus clarkii, [...] Read more.

Macrobrachium rosenbergii is a valuable freshwater prawn in Asian aquaculture. In recent years, a new symptom that was generally called “white head” has caused high mortality in M. rosenbergii farms in China. Samples of M. rosenbergii, M. nipponense, Procambarus clarkii, M. superbum, Penaeus vannamei, and Cladocera from a farm suffering from white head in Jiangsu Province were collected and analyzed in this study. Pathogen detection showed that all samples were positive for Decapod iridescent virus 1 (DIV1). Histopathological examination revealed dark eosinophilic inclusions and pyknosis in hematopoietic tissue, hepatopancreas, and gills of M. rosenbergii and M. nipponense. Blue signals of in situ digoxigenin-labeled loop-mediated isothermal amplification appeared in hematopoietic tissue, hemocytes, hepatopancreatic sinus, and antennal gland. Transmission electron microscopy of ultrathin sections showed a large number of DIV1 particles with a mean diameter about 157.9 nm. The virogenic stromata and budding virions were observed in hematopoietic cells. Quantitative detection with TaqMan probe based real-time PCR of different tissues in naturally infected M. rosenbergii showed that hematopoietic tissue contained the highest DIV1 load with a relative abundance of 25.4 ± 16.9%. Hepatopancreas and muscle contained the lowest DIV1 loads with relative abundances of 2.44 ± 1.24% and 2.44 ± 2.16%, respectively. The above results verified that DIV1 is the pathogen causing white head in M. rosenbergii. M. nipponense and Pr. clarkii are also species susceptible to DIV1. Full article

(This article belongs to the Special Issue Family Iridoviridae: Molecular and Ecological Studies of a Family Infecting Invertebrates and Ectothermic Vertebrates)

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Figure 1
Clinical symptoms of M. rosenbergii (20180620) naturally infected with DIV1. (A) Overall appearance of a diseased prawn in water. (B) Close-up of cephalothoraxes. Blue arrows show white area under the cuticle at the base of rostrum. White arrows indicate hepatopancreas atrophy, color fading, and yellowing. Full article ">Figure 2
Histopathological features of Davidson’s alcohol-formalin-acetic acid fixative (DAFA) fixed M. rosenbergii (A–C) and M. nipponense (D) samples 20180620. White arrows show the eosinophilic inclusions and black arrows show the karyopyknotic nuclei. (A) Hematoxylin and eosin (H&E) staining of the hematopoietic tissue. (B,D) H&E staining of hepatopancreas. (C) H&E staining of gills. Bar, 20 μm (A,B), 50 μm (C), and 10 μm (D), respectively. Full article ">Figure 3
In situ digoxigenin-labeled loop-mediated isothermal amplification (ISDL) targeting the gene of the second largest subunit of DNA-directed RNA polymerase II of DIV1 on histological sections of M. rosenbergii (A–F), M. nipponense (G), and Pr. clarkii (H,I) samples 20180620. (A,H) Hematopoietic tissue; (B,D,G,I) hepatopancreas; (C) gills; (E) antennal gland; (F) ovaries. In (A–C,H), blue signals were observed in hematopoietic tissue, hemocytes in the sinus of the hepatopancreas, and in gills. In (D,G,I), blue signals exist in some hepatopancreatic R-cells and myoepithelial fibers. In (E), blue signals exist in the coelomosac epithelium. In (F), blue signals exist in the epithelium. Bar, 20 µm (A,D–F,I), 50 µm (B,C), and 10 µm (G,H), respectively. Full article ">Figure 4
TEM of hematopoietic tissue of naturally infected M. rosenbergii samples 20180620. (A) A large numbers of virions in hematopoietic tissue. (B) DIV1 budded and acquired an envelope from the plasma membrane. (C) DIV1 replication and assembly in hematopoietic cells. (D) The stages of nucleocapsid assembly, which are indicated with numbers 1–3, and a complete nucleocapsid is indicated with number 4. The capsids at stage 2 and 3 should have a small opening at one vertex but may not be visible in the picture due to the ultrathin section. (E) Crescent-shaped structures. (F–I) As the assembling process continues, the crescent-shaped structure curves to form icosahedral capsids. (J) A mature virion with a dense core was eventually formed. N: nucleus; *: a large electron-lucent virogenic stroma; white arrows: paracrystalline array of viral particles; black arrows: budding virions; and white triangles: budded virions that acquired an envelope. Full article ">Figure 5
Relative abundance of DIV1 for different tissues of fifteen DIV1-infected M. rosenbergii samples. Columns without sharing of a same letter indicate significant difference of p < 0.05; columns without a same color indicate a highly significant difference of p < 0.01. Full article ">

14 pages, 6251 KiB

Open AccessArticle

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

by Eva-Stina Isabella Edholm, Francisco De Jesús Andino, Jinyeong Yim, Katherine Woo and Jacques Robert

Viruses 2019, 11(4), 330; https://doi.org/10.3390/v11040330 - 6 Apr 2019

Cited by 11 | Viewed by 3077

Abstract

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 Xenopus laevis critically rely on MHC class I-like (mhc1b10.1.L or XNC10)-restricted innate-like (i)T cells (iVα6 T [...] Read more.

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 Xenopus laevis 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 iVα6 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. Full article

(This article belongs to the Special Issue Family Iridoviridae: Molecular and Ecological Studies of a Family Infecting Invertebrates and Ectothermic Vertebrates)

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15 pages, 2455 KiB

Open AccessArticle

Geographic Distribution of Epizootic haematopoietic necrosis virus (EHNV) in Freshwater Fish in South Eastern Australia: Lost Opportunity for a Notifiable Pathogen to Expand Its Geographic Range

by Joy A. Becker, Dean Gilligan, Martin Asmus, Alison Tweedie and Richard J. Whittington

Viruses 2019, 11(4), 315; https://doi.org/10.3390/v11040315 - 1 Apr 2019

Cited by 3 | Viewed by 3718

Abstract

Epizootic haematopoietic necrosis virus (EHNV) was originally detected in Victoria, Australia in 1984. It spread rapidly over two decades with epidemic mortality events in wild redfin perch (Perca fluviatilis) and mild disease in farmed rainbow trout (Oncorhynchus mykiss) being [...] Read more.

Epizootic haematopoietic necrosis virus (EHNV) was originally detected in Victoria, Australia in 1984. It spread rapidly over two decades with epidemic mortality events in wild redfin perch (Perca fluviatilis) and mild disease in farmed rainbow trout (Oncorhynchus mykiss) being documented across southeastern Australia in New South Wales (NSW), the Australian Capital Territory (ACT), Victoria, and South Australia. We conducted a survey for EHNV between July 2007 and June 2011. The disease occurred in juvenile redfin perch in ACT in December 2008, and in NSW in December 2009 and December 2010. Based on testing 3622 tissue and 492 blood samples collected from fish across southeastern Australia, it was concluded that EHNV was most likely absent from redfin perch outside the endemic area in the upper Murrumbidgee River catchment in the Murray–Darling Basin (MDB), and it was not detected in other fish species. The frequency of outbreaks in redfin perch has diminished over time, and there have been no reports since 2012. As the disease is notifiable and a range of fish species are known to be susceptible to EHNV, existing policies to reduce the likelihood of spreading out of the endemic area are justified. Full article

(This article belongs to the Special Issue Family Iridoviridae: Molecular and Ecological Studies of a Family Infecting Invertebrates and Ectothermic Vertebrates)

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15 pages, 7414 KiB

Open AccessArticle

Pathogen Risk Analysis for Wild Amphibian Populations Following the First Report of a Ranavirus Outbreak in Farmed American Bullfrogs (Lithobates catesbeianus) from Northern Mexico

by Bernardo Saucedo, José M. Serrano, Mónica Jacinto-Maldonado, Rob S. E. W. Leuven, Abraham A. Rocha García, Adriana Méndez Bernal, Andrea Gröne, Steven J. Van Beurden and César M. Escobedo-Bonilla

Viruses 2019, 11(1), 26; https://doi.org/10.3390/v11010026 - 3 Jan 2019

Cited by 15 | Viewed by 5268

Abstract

Ranaviruses are the second deadliest pathogens for amphibian populations throughout the world. Despite their wide distribution in America, these viruses have never been reported in Mexico, the country with the fifth highest amphibian diversity in the world. This paper is the first to [...] Read more.

Ranaviruses are the second deadliest pathogens for amphibian populations throughout the world. Despite their wide distribution in America, these viruses have never been reported in Mexico, the country with the fifth highest amphibian diversity in the world. This paper is the first to address an outbreak of ranavirus in captive American bullfrogs (Lithobates catesbeianus) from Sinaloa, Mexico. The farm experienced high mortality in an undetermined number of juveniles and sub-adult bullfrogs. Affected animals displayed clinical signs and gross lesions such as lethargy, edema, skin ulcers, and hemorrhages consistent with ranavirus infection. The main microscopic lesions included mild renal tubular necrosis and moderate congestion in several organs. Immunohistochemical analyses revealed scant infected hepatocytes and renal tubular epithelial cells. Phylogenetic analysis of five partial ranavirus genes showed that the causative agent clustered within the Frog virus 3 clade. Risk assessment with the Pandora⁺ protocol demonstrated a high risk for the pathogen to affect amphibians from neighboring regions (overall Pandora risk score: 0.619). Given the risk of American bullfrogs escaping and spreading the disease to wild amphibians, efforts should focus on implementing effective containment strategies and surveillance programs for ranavirus at facilities undertaking intensive farming of amphibians. Full article

(This article belongs to the Special Issue Family Iridoviridae: Molecular and Ecological Studies of a Family Infecting Invertebrates and Ectothermic Vertebrates)

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Figure 1
Location of city of the outbreak in Mexico. The city of Guasave is indicated by a black circle within the province of Sinaloa (grey). Mexico City, the place from which the bullfrogs were imported from, is shown in black. Full article ">Figure 2
Macroscopic lesions of ranavirus-infected bullfrogs. (A) Extensive ulceration of the forelimb. (B) Extensive hepatic necrosis and epicardial pallor and hemorrhages (black arrow). Full article ">Figure 3
Histopathology of ranavirus-infected American bullfrogs. (A) Liver section from an affected bullfrog with mild congestion of sinusoids. (B) Kidney section shows an area of mild tubular necrosis characterized by cytoplasmic hyper-eosinophilia, loss of nuclei, and mild nuclear pyknosis (black square). Intraluminal protein casts are also seen (black arrows). Hematoxylin/Eosin staining. Magnification for all photos 20×/100 μm. Full article ">Figure 4
Immunohistochemistry of ranavirus-infected American bullfrogs (Lithobates catesbeianus). (A) Immunohistochemical staining of kidney, in which positive immunolabelling (red staining) is present in the renal cortical interstitium along with areas of necrosis and scant inflammation. (B) Immunohistochemical staining of a liver from an affected bullfrog with a focal area of positive immunolabelling (red staining) in the cytoplasm of a Kupffer cell. (C) Serial section of the kidney from the same animal without secondary antibody (control), (D) Serial section of liver from the same animal without secondary antibody (control). (E) Histological kidney section from an anuran which was PCR negative for ranavirus infection (control). (F) Histological liver section from an anuran which was PCR negative for ranavirus infection (control). Magnification for all photos 40×/50 μm. Full article ">Figure 5
PCR products of five ranavirus genes. All products of the Mexican virus samples (S) are of similar size to those of the FV3 virus positive control (P). Abbreviations: W (water), S (Mexican virus sample), P (Positive control frog virus 3 isolate Oophaga pumilio/2015/Netherlands/UU3150324001 (GenBank no. MF360246)). 100 bp ladder. Full article ">Figure 6
Phylogeny of five partial ranavirus gene sequences (1000 bootstrap values). The Mexican isolate FV3 L. catesbeianus (underlined) clusters closely within the FV3 ranavirus clade. Isolates and Genbank numbers used: Frog virus 3/Lithobates catesbeianus/2018/Mexico (GenBank accession numbers for partial sequences available in <a href="#app1-viruses-11-00026" class="html-app">Supplementary Table S1</a>), frog virus 3 (AY548484), frog virus 3 isolate SMME (KJ175144), tortoise ranavirus isolate 1 (882/96) (KP266743), Bosca’s newt virus isolate GA11001 (KJ703118), frog virus 3 isolate Oophaga pumilio/2015/Netherlands/UU3150324001 (MF360246), common midwife toad ranavirus isolate Mesotriton alpestris/2008/E (JQ231222), common midwife toad virus isolate P11114 (KJ703146), Testudo hermanni ranavirus isolate CH8/96 (KP266741), tiger frog virus (AF389451), soft-shelled turtle iridovirus (EU627010), European sheatfish virus (JQ724856), epizootic hematopoietic necrosis virus (FJ433873), Ambystoma tigrinum stebbensi virus (AY150217), Andrias davidianus ranavirus isolate (KF033124), Andrias davidianus ranavirus isolate 1201 (KC865735), Chinese giant salamander iridovirus, isolate CGSIV-HN1104, (KF512820), and Rana grylio virus (JQ654586). Only bootstrap values higher than 50 are shown. Full article ">

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15 pages, 1053 KiB

Open AccessTechnical Note

eDNA Increases the Detectability of Ranavirus Infection in an Alpine Amphibian Population

by Claude Miaud, Véronique Arnal, Marie Poulain, Alice Valentini and Tony Dejean

Viruses 2019, 11(6), 526; https://doi.org/10.3390/v11060526 - 6 Jun 2019

Cited by 33 | Viewed by 5431

Abstract

The early detection and identification of pathogenic microorganisms is essential in order to deploy appropriate mitigation measures. Viruses in the Iridoviridae family, such as those in the Ranavirus genus, can infect amphibian species without resulting in mortality or clinical signs, and they can [...] Read more.

The early detection and identification of pathogenic microorganisms is essential in order to deploy appropriate mitigation measures. Viruses in the Iridoviridae family, such as those in the Ranavirus genus, can infect amphibian species without resulting in mortality or clinical signs, and they can also infect other hosts than amphibian species. Diagnostic techniques allowing the detection of the pathogen outside the period of host die-off would thus be of particular use. In this study, we tested a method using environmental DNA (eDNA) on a population of common frogs (Rana temporaria) known to be affected by a Ranavirus in the southern Alps in France. In six sampling sessions between June and September (the species’ activity period), we collected tissue samples from dead and live frogs (adults and tadpoles), as well as insects (aquatic and terrestrial), sediment, and water. At the beginning of the breeding season in June, one adult was found dead; at the end of July, a mass mortality of tadpoles was observed. The viral DNA was detected in both adults and tadpoles (dead or alive) and in water samples, but it was not detected in insects or sediment. In live frog specimens, the virus was detected from June to September and in water samples from August to September. Dead tadpoles that tested positive for Ranavirus were observed only on one date (at the end of July). Our results indicate that eDNA can be an effective alternative to tissue/specimen sampling and can detect Ranavirus presence outside die-offs. Another advantage is that the collection of water samples can be performed by most field technicians. This study confirms that the use of eDNA can increase the performance and accuracy of wildlife health status monitoring and thus contribute to more effective surveillance programs. Full article

(This article belongs to the Special Issue Family Iridoviridae: Molecular and Ecological Studies of a Family Infecting Invertebrates and Ectothermic Vertebrates)

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8 pages, 1821 KiB

Open AccessBrief Report

Localization of Frog Virus 3 Conserved Viral Proteins 88R, 91R, and 94L

by Emily Penny and Craig R. Brunetti

Viruses 2019, 11(3), 276; https://doi.org/10.3390/v11030276 - 19 Mar 2019

Cited by 2 | Viewed by 2861

Abstract

The characterization of the function of conserved viral genes is central to developing a greater understanding of important aspects of viral replication or pathogenesis. A comparative genomic analysis of the iridoviral genomes identified 26 core genes conserved across the family Iridoviridae. Three [...] Read more.

The characterization of the function of conserved viral genes is central to developing a greater understanding of important aspects of viral replication or pathogenesis. A comparative genomic analysis of the iridoviral genomes identified 26 core genes conserved across the family Iridoviridae. Three of those conserved genes have no defined function; these include the homologs of frog virus 3 (FV3) open reading frames (ORFs) 88R, 91R, and 94L. Conserved viral genes that have been previously identified are known to participate in a number of viral activities including: transcriptional regulation, DNA replication/repair/modification/processing, protein modification, and viral structural proteins. To begin to characterize the conserved FV3 ORFs 88R, 91R, and 94L, we cloned the genes and determined their intracellular localization. We demonstrated that 88R localizes to the cytoplasm of the cell while 91R localizes to the nucleus and 94L localizes to the endoplasmic reticulum (ER). Full article

(This article belongs to the Special Issue Family Iridoviridae: Molecular and Ecological Studies of a Family Infecting Invertebrates and Ectothermic Vertebrates)

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