Amphibian Virus Research Articles

Marked systemic necrotizing disease in a Leptodactylus vastus (Anura: Leptodactylidae) from an urban reserve in northeastern Brazil

The decline of amphibian populations concerns scientists worldwide and has been related to the spreading infectious diseases, among other environmental factors. Acute mass mortality with hemorrhagic and necrotic lesions has been described and associated with wild amphibian viruses. These diseases were first recorded in Central America and Australia in the early 1990s and later in Brazilian Atlantic Forest fragments. Epizootiological data on these infections must be included in other Brazilian phytogeographic regions and domains. This work aimed to report a systemic necrotic morbid state in a native Leptodactylus vastus from an urban reserve in northeastern Brazil, describing clinical and pathological findings and discussing its importance and likely etiologies. An adult L. vastus presented an erythematous oral cavity with multifocal point-like erosions, sublingual congested vessels, oropharyngeal edema, and mild esophageal mucosal prolapse. After therapeutic protocol, the patient showed no improvement; then, he was humanely euthanized. Necropsy demonstrated moderate to marked multicentric chronic necrotizing lesions. Histopathology revealed diffuse necrotic tissues with a few intracytoplasmic amphophilic inclusions in cellular remnants, with minor hemoparasites, helminths, and bacterial granulomatous coinfections. Clinical and pathological findings suggested a major chronic viral infection with minor coinfections. In the current case, confirmation of etiology struggled with financial and logistic limitations. Future partnerships will enable definitive diagnostics. This is the first record of systemic necrotizing disease in a wild frog in Ceará state, northeastern Brazil, and it is a red flag to emerging infectious diseases surveillance in this geographic area.

Revealing the uncharacterised diversity of amphibian and reptile viruses

Amphibians and non-avian reptiles represent a significant proportion of terrestrial vertebrates, however knowledge of their viruses is not proportional to their abundance. Many amphibians and reptiles have strict habitual environments and localised populations and are vulnerable to viral outbreaks and potential elimination as a result. We sought to identify viruses that were hidden in amphibian and reptile metatranscriptomic data by screening 235 RNA-sequencing datasets from a 122 species covering 25 countries. We identified 26 novel viruses and eight previously characterised viruses from fifteen different viral families. Twenty-five viruses had RNA genomes with identity to Arteriviridae, Tobaniviridae, Hantaviridae, Rhabdoviridae, Astroviridae, Arenaviridae, Hepeviridae, Picornaviridae, Orthomyxoviridae, Reoviridae, Flaviviridae and Caliciviridae. In addition to RNA viruses, we also screened datasets for DNA viral transcripts, which are commonly excluded from transcriptomic analysis. We identified ten DNA viruses with identity to Papillomaviridae, Parvoviridae, Circoviridae and Adomaviridae. With the addition of these viruses, we expand the global amphibian and reptile virome and identify new potentially pathogenic viruses that could challenge populations. We speculate that amphibian viruses often have simpler genomes than those in amniotes, as in the case of the Secondpapillomavirinae and Orthomyxoviridae viruses identified in this study. In addition, we find evidence of inter-family recombination in RNA viruses, and we also identify new members of the recombinant Adomaviridae family. Overall, we provide insights into the uncharacterised diversity of amphibian and reptile viruses with the aim of improving population management, treatment and conservation into the future.

Comparative Genomics of an Emerging Amphibian Virus.

Ranaviruses, a genus of the Iridoviridae, are large double-stranded DNA viruses that infect cold-blooded vertebrates worldwide. Ranaviruses have caused severe epizootics in commercial frog and fish populations, and are currently classified as notifiable pathogens in international trade. Previous work shows that a ranavirus that infects tiger salamanders throughout Western North America (Ambystoma tigrinum virus, or ATV) is in high prevalence among salamanders in the fishing bait trade. Bait ATV strains have elevated virulence and are transported long distances by humans, providing widespread opportunities for pathogen pollution. We sequenced the genomes of 15 strains of ATV collected from tiger salamanders across western North America and performed phylogenetic and population genomic analyses and tests for recombination. We find that ATV forms a monophyletic clade within the rest of the Ranaviruses and that it likely emerged within the last several thousand years, before human activities influenced its spread. We also identify several genes under strong positive selection, some of which appear to be involved in viral virulence and/or host immune evasion. In addition, we provide support for the pathogen pollution hypothesis with evidence of recombination among ATV strains, and potential bait-endemic strain recombination.

Molecular characterization of iridoviruses isolated from sympatric amphibians and fish

Iridoviruses infect invertebrates (primarily insects and crustaceans) and ectothermic vertebrates (fish, amphibians, and reptiles). Identical, or nearly identical viruses, have been isolated from different animals within the same taxonomic class, indicating that infection by a given virus is not limited to a single species. Although inter-class infections have been documented following experimental infection with vertebrate iridoviruses, it is not clear whether such infections occur in nature. Here we report the isolation of apparently identical iridoviruses from wild sympatric fish (the threespine stickleback, Gasterostelus aculeatus) and amphibians (the red-legged frog, Rana aurora). Viruses isolated from sticklebacks (stickleback virus, SBV) and from a red-legged frog tadpole (tadpole virus 2, TV2) replicated in fathead minnow (FHM) cells and synthesized proteins which co-migrated with those of frog virus 3 (FV3). Following restriction endonuclease digestion of viral DNA with Hind III and Xba I, gel analysis showed that the profiles of SBV and TV2 were identical to each other and distinct from FV3. Using oligonucleotide primers specific for a highly conserved region of the iridovirus major capsid protein, an ∼500 nucleotide DNA fragment was amplified from SBV and TV2. Sequence analysis showed that within this 500 nucleotide region SBV and TV2 were identical to each other and to FV3. Taken together these results provide the first evidence that iridoviruses naturally infect animals belonging to different taxonomic classes, and strengthen the suggestion that fish may serve as a reservoir for amphibian viruses or vice versa.

Detection of antibodies against iridoviruses in the serum of the amphibian Bufo marinus

Sera from the amphibian Bufo marinus (cane or marine toad) were investigated using a newly-developed enzyme-linked immunosorbent assay for the detection of antibodies to ranaviruses (Family Iridoviridae). Epizootic haematopoietic necrosis virus (EHNV) or Bohle iridovirus (BIV) was affinity purified from cell culture supernatants and simultaneously bound to the solid phase using specific linker antibodies. After binding to antigen, antibodies in Bufo marinus serum were then detected with a specific anti-immunoglobulin reagent. Of 21 Bufo marinus sera, 3 contained antibodies against EHNV, BIV or related viruses in the ranavirus group, at titres greater than 3200. Reactivity of cane toad antibodies against BIV, an amphibian virus, was greater than that against similar concentrations of EHNV, a piscine virus.

Amphibian and Piscine Iridoviruses Proposal for Nomenclature and Taxonomy Based on Molecular and Biological Properties

We have compared a number of properties of the well-characterized iridovirus, frog virus 3, with two other iridoviruses from amphibia, bullfrog edema virus and Lucké triturus virus, and with a piscine iridovirus, goldfish virus (GFV), to provide information for developing taxonomic classification of these viruses and establishing their ecological niche. Purified virions had similar size and shape (icosahedral) for each virus, and the genomic DNAs of each virus were methylated by a virus-induced DNA methyltransferase. The three amphibian viruses replicated equally well in fish (FHM), hamster (BHK), and human (WI-38) cell monolayer with identical cytopathology, while GFV failed to replicate in these cell lines. However, GFV replicated albeit at a slow rate, in a goldfish cell line; there was no detectable replication by the amphibian viruses in these cells. The amphibian iridoviruses had virtually similar DNA sequences, while those of GFV were markedly different. Analyses of virus-induced polypeptides in infected cells corroborated the DNA analyses; the polypeptides of the amphibian viruses were similar and distinct from those of the fish virus. Nongenetic reactivation could only be accomplished between the three amphibian viruses but not with the piscine virus. Based on these data, we suggest taxonomic and nomenclature designations of amphibian and piscine iridoviruses.

Fish cell culture: two newly developed cell lines from Atlantic sturgeon (Acipenser oxyrhynchus) and guppy (Poecilia reticulata)

Two new cell cultures from fish, SH cells from cardiac tissue of a mature Atlantic sturgeon (Acipenser oxyrhynchus) and GFT cells from fully formed unborn embryos of a female guppy (Poecilia reticulata), were established through a series of subcultivations. The SH cells (sturgeon heart) are fibroblastlike and the GFT cells (guppy fetal tissue) are epithelioid in morphology. The SH cell line was started in 1977 and the GFT line in 1979, and both are past their 30th passage. Maximum growth, after 15 days, was observed at 18 °C for both cell lines. They require 3–5% extra salt for optimum growth. Generally, GFT cells have higher relative plating efficiency and a shorter generation time than SH cells. Sensitivity of both SH and GFT cells to viral infection was tested using an amphibian virus (LT-1V) and a fish virus (IPNV). Cytopathic changes, of the cells experimentally infected by these viruses were evaluated. A low sensitivity of SH cells and GFT cells was found in response to IPNV and LT-1V, respectively. Electron microscopy revealed that both viruses appeared to have different sites of replication in the infected cell lines. Metaphase plates of GFT cells indicated prominent metacentric and submetacentric chromosomes. The chromosomes of SH cells consisted of both micro- and macro-chromosomes. At least three isozymes (lactic acid dehydrogenase, malate dehydrogenase, and phosphohexose isomerase) were found to be suitable for distinguishing among GFT, SH, and lumpfish cell lines.

Macromolecular synthesis in cells infected by frog virus 3.

The best studied member of the Iridoviridae is frog virus 3 (FV3), isolated by Granoff et al. (1966) from a renal adenocarcinoma of the leopard frog, Rana pipiens. Similar isolates were made from normal frog kidney and liver (FV1, FY 2, FV5–FY24), but FV3 was chosen for further investigation because of its presence in the tumor. As it turned out, this unusual cytoplasmic DNA virus bore no causal or even helper relationship to the tumor, which was the result of infection with the Lucké herpesvirus (Naegele et al. 1974), but it did have a number of interesting characteristics that made it a worthy object of study in its own right. FV3 is probably similar or identical to other amphibian virus isolates obtained from bullfrogs, newts, and toads (Wolf et al. 1968; Clark et al. 1969).KeywordsLeopard FrogMacromolecular SynthesisFrog VirusLate MessageHost Protein SynthesisThese keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

Fish Cell Culture: Characteristics of a Continuous Fibroblastic Cell Line from Walleye (Stizostedion vitreum vitreum)

A cell line (WC-1) was initiated from a dermal tumor from walleye (Stizostedion vitreum vitreum). The fibroblastic cells were propagated in Eagle's minimum essential medium, supplemented with 10% fetal bovine serum, as monolayer cultures that exhibited only slight density-dependent inhibition of mitosis. Cells, which were characterized at passage levels 45–46, grew optimally at 20 to 25 °C, but growth was limited at 10 and 30 °C. The line has a chromosome modal number of 48 or 49. Zymograms for lactate dehydrogenase (LDH) and malate dehydrogenase (MDH) of WC-1 cells and of normal walleye tissue were very similar, indicating that the WC-1 line may be distinguished by these isozymes. Cell cultures supported replication (with resulting cytopathology) of infectious pancreatic necrosis virus (IPNV, strain VR 299) and the amphibian icosahedral cytoplasmic deoxyribovirus LT-1. The WC-1 line which has now been subcultured more than 100 times can properly be regarded as a continuous cell line.Key words: poikilotherm vertebrate cell line, fish virus, amphibian virus, neoplasm, walleye dermal sarcoma, lactate dehydrogenase, malate dehydrogenase, isozyme

New Cell Line from the Marine Lumpfish, Cyclopterus lumpus

A cell line was grown from fin tissue of lumpfish (Cyclopterus lumpus). The cells were propagated in RTO medium supplemented with NaCl at 18 °C and have a generation time of 108 h. They are fibroblast-like, contain numerous cytoplasmic granules, are susceptible to amphibian virus (LT-1), and have an average chromosome number of 2N = 50.

Antiviral activity of aqueous clam (Mya arenaria) extract on amphibian virus (LT-1).

An aqueous extract of the soft-shell clam displayed antiviral activity which protected against infection of fathead minnow (FHM) cells by the amphibian virus (LT-1). The extract did not inactivate the virus particle extracellularly, nor could a combined action on virus and cell receptor sites account for the inhibitory effect of the extract. The observed inhibition appeared to be at the intracellular level.

Observations on rainbow trout ovary cells infected with amphibian virus (LT-1)

The amphibian virus, LT-1, produced relatively irregular necrotic areas in the monolayer of rainbow trout ovarian cells. The infected cells underwent the following cytopathic changes: extensive cytoplasmic vacuolation, formation of multiple nuclei, and paranuclear cytoplasmic inclusions.The site of replication of the virus in the infected rainbow trout ovarian cells was demonstrated to be located at the less electron-dense areas of the cytoplasm and surrounded by numerous vacuoles. A comparison of the susceptibility to LT-1 virus of rainbow trout ovarian cells with that of fathead minnow cells resulted in a calculated cytopathic index of 4.48.

Thermoelectrically cooled temperature-gradient apparatus for comparative cell and virus temperature studies.

Establishment of a near-linear temperature gradient in an incubator has been accomplished by the application of heat to one terminus of a conducting body, normally a metal bar, and the removal of heat from the other terminus of the conducting body. Such incubators have been complex and unwieldy because of the need for mechanical refrigeration. We have described a simplified temperature gradient incubator which uses thermoelectric module cooling coupled with electric heating. Along the gradient, 20 stations in two parallel rows of 10, each accommodating a 30-ml plastic cell culture flask, were continually monitored by an electronic thermometer, and the temperatures were recorded. By manipulation of two simple potentiometer controls, any temperature gradient between 0 and 50 C could be obtained. Minor deviations which occurred between theoretically perfect and obtained temperature gradients were reproducible and readily measured. The gradient incubator was particularly applicable to (i) simultaneously studying a given biological activity over the entire temperature range supporting the growth of a given cell, virus, or microorganism, or (ii) precisely defining the upper or lower temperature limits of a biological system by 10-point determinations. Preliminary experiments have demonstrated the usefulness of the apparatus in characterizing the temperature limits for growth in vitro of cells of reptilian cell lines. The gradient incubator was also successfully utilized for the characterization of the effect of temperature on the efficiency of plating of amphibian viruses and possible temperature variants of those viruses.

Thermoelectrically Cooled Temperature-Gradient Apparatus for Comparative Cell and Virus Temperature Studies

Establishment of a near-linear temperature gradient in an incubator has been accomplished by the application of heat to one terminus of a conducting body, normally a metal bar, and the removal of heat from the other terminus of the conducting body. Such incubators have been complex and unwieldy because of the need for mechanical refrigeration. We have described a simplified temperature gradient incubator which uses thermoelectric module cooling coupled with electric heating. Along the gradient, 20 stations in two parallel rows of 10, each accommodating a 30-ml plastic cell culture flask, were continually monitored by an electronic thermometer, and the temperatures were recorded. By manipulation of two simple potentiometer controls, any temperature gradient between 0 and 50 C could be obtained. Minor deviations which occurred between theoretically perfect and obtained temperature gradients were reproducible and readily measured. The gradient incubator was particularly applicable to (i) simultaneously studying a given biological activity over the entire temperature range supporting the growth of a given cell, virus, or microorganism, or (ii) precisely defining the upper or lower temperature limits of a biological system by 10-point determinations. Preliminary experiments have demonstrated the usefulness of the apparatus in characterizing the temperature limits for growth in vitro of cells of reptilian cell lines. The gradient incubator was also successfully utilized for the characterization of the effect of temperature on the efficiency of plating of amphibian viruses and possible temperature variants of those viruses.

Comparative Immune Response to Amphibian Cytoplasmic Viruses Assayed by the Complement-Fixation and Gel Immunodiffusion Tests

Abstract Rabbits and guinea pigs immunized with several amphibian cytoplasmic virus strains consistently responded with neutralizing, complement-fixing, and precipitating virus-specific antibodies. Complement-fixation and gel-immunodiffusion tests revealed a close antigenic relationship among all strains tested, in agreement with previous results obtained with the virus-neutralization test. No antigenic relationship was demonstrated between the amphibian viruses and the morphologically similar Tipula iridescent virus. Bullfrogs and snapping turtles responded irregularly to amphibian virus inoculation with low-titered neutralizing and/or precipitating antibody. However, antibody was detected in pre-immunization sera of some frogs and turtles, suggesting prior exposure to related viruses. Viral antigen was not detected in leopard frog renal tumors by the immunodiffusion or complement-fixation tests. Similarly, antibody to cytoplasmic virus antigen could not be detected in sera of tumor-bearing or normal leopard frogs.

Factors Affecting Rainbow Trout Ovary Cells Cultivated In vitro

The rainbow trout ovary cells proliferated well at 18 and 23 C, and at culture medium pH value of 7.1 and 7.9. Outside these limits cell proliferation was hindered and the cellular morphology was affected significantly. The ovary cells have been shown to be susceptible to amphibian virus (LT-1).

A serological study of polyhedral cytoplasmic viruses isolated from amphibia

Antisera to FV3, a frog virus of the cytoplasmic polyhedral type, were prepared in rabbits and tested for virus-neutralization and precipitating activity with FV3 and eight other isolates of amphibian viruses of the same morphological type (FV1, FV13, FV16; LT1, LT2, LT3, L4b, and TEV). After appropriate absorption, two strong precipitation bands were formed in Ouchterlony tests with viral antigens prepared from ether-treated extracts of FV3-infected cultures; both bands gave reactions of identity with all nine virus isolates. Rabbit anti FV3 serum had similar neutralizing activity against the four FV viruses and LT1 and TEV virus isolates. Thus the nine isolates were not distinguishable either by virus-neutralization or by immunodiffusion tests with these antisera. In addition, FV1, FV3, LT1 and TEV produced a lethal infection in young Fowler's toads, the gross pathology being similar in each case. The results suggest that these isolates of polyhedral viruses are closely related or represent a single agent which has been obtained from various species in different geographical locations.

Temperature optima of mammalian and amphibian viruses in cell cultures of homeothermic and poikilothermic origin.

The effect of incubation temperature on the growth of the mammalian viruses HSV and vaccinia and the amphibian viruses FV-1 and LT-1 was studied in cell cultures of mammalian, avian and reptilian origin. Unlike the mammalian (monkey) and avian (chick) cells, cells of the reptilian cell line TH-1A multiplied at optimal rates at 23°C and 30°C but did not multiply at 36° C. Despite the different temperature optima of the host cells studied, the mammalian viruses replicated at maximum rates at 36° C and the amphibian viruses replicated at maximum rates at 23° C in each host cell type. At the intermediate temperature of 30° C near maximal rates of replication of both amphibian and mammalian viruses were usually observed. However, at this temperature the turtle cell line TH-1A supported reduced rates of replication of both the “warm optimum” vaccinia virus and the“cool optimum” LT-1 and FV-1 viruses. The results suggest that the viral genome is the major determinant of the temperature required for synthesis of a given virus, but the host cell may influence the finer limits of the temperature range supporting replication.

Electron Microscopic Observations On An Amphibian Polyhedral Virus (LT-1) Propagated in Vitro in Reptilian Cells

Polyhedral, cytoplasmically derived, viral agents have been isolated from Rana pipiens and characterized in baby hamster kidney and Fathead minnow cells in vitro. These viruses are not similar to the Herpes-type virus associated with the Lucké tumor in these frogs. Clark, et al have recently described additional isolates of similar polyhedral agents from the leopard frog after passage through red efts (Triturus viridescens). This virus series, designated LT-1, -2, -3, -4, has been propagated in the turtle heart cell line TH-1. This report presents morphological studies on these isolates.Slow speed centrifuged cell packs were fixed in cold chrome-osmium (Dalton) followed by fixation in cold 0.5% aqueous uranyl-acetate with 0.54% sucrose. Sections were double stained with uranyl-acetate and lead citrate.The virus particles were observed to form in juxta-nuclear viroplasmic regions within the cell cytoplasm. Evidence of intranuclear virus synthesis has not been observed, to date. Viroplasmic regions were frequently surrounded by mitochondria. Within the viroplasmic regions the fixation used preserved a filamentous mesh work which is observed to be continuous with that which is accumulated inside the virions (Figs. 1, 2). This filamentous material is similar to that observed in Mycoplasma and in poxvirus viroplasm.

Tadpole edema virus: a viscerotropic pathogen for anuran amphibians.

Journal Article Tadpole Edema Virus: A Viscerotropic Pathogen for Anuran Amphibians Get access Ken Wolf, Ken Wolf From the Bureau of Sport Fisheries and Wildlife, Eastern Fish Disease Laboratory, Kearneysville, West Virginia 25430 Search for other works by this author on: Oxford Academic PubMed Google Scholar G. L. Bullock, G. L. Bullock From the Bureau of Sport Fisheries and Wildlife, Eastern Fish Disease Laboratory, Kearneysville, West Virginia 25430 Search for other works by this author on: Oxford Academic PubMed Google Scholar C. E. Dunbar, C. E. Dunbar From the Bureau of Sport Fisheries and Wildlife, Eastern Fish Disease Laboratory, Kearneysville, West Virginia 25430 Search for other works by this author on: Oxford Academic PubMed Google Scholar M. C. Quimby M. C. Quimby From the Bureau of Sport Fisheries and Wildlife, Eastern Fish Disease Laboratory, Kearneysville, West Virginia 25430 Search for other works by this author on: Oxford Academic PubMed Google Scholar The Journal of Infectious Diseases, Volume 118, Issue 3, June 1968, Pages 253–262, https://doi.org/10.1093/infdis/118.3.253 Published: 01 June 1968 Article history Received: 30 September 1967 Published: 01 June 1968