Characterization of ranaviruses isolated from lumpfish Cyclopterus lumpus L. in the North Atlantic area: proposal for a new ranavirus species (European North Atlantic Ranavirus).

The commercial production of lumpfish Cyclopterus lumpus L. is expanding with the increased demand for their use as cleaner fish, to control sea-lice numbers, at marine Atlantic salmon Salmo salar L. aquaculture sites throughout Northern Europe. A new ranavirus has been isolated from lumpfish at multiple locations in the North Atlantic area. First isolated in 2014 in the Faroe Islands, the virus has subsequently been found in lumpfish from Iceland in 2015 and from Scotland and Ireland in 2016. The Icelandic lumpfish ranavirus has been characterized by immunofluorescent antibody test, optimal growth conditions and transmission electron microscopy. Partial sequences of the major capsid protein gene from 12 isolates showed 99.79-100% nt identity between the lumpfish ranaviruses. Complete genome sequencing from three of the isolates and phylogenetic analysis based on the concatenated 26 iridovirus core genes suggest these lumpfish ranavirus isolates form a distinct clade with ranaviruses from cod Gadus morhua L. and turbot Scophthalmus maximus L. isolated in Denmark in 1979 and 1999, respectively. These data suggest that these viruses should be grouped together as a new ranavirus species, European North Atlantic Ranavirus, which encompasses ranaviruses isolated from marine fishes in European North Atlantic waters.

Isolation and characterization of a ranavirus from koi, Cyprinus carpio L., experiencing mass mortalities in India.

We investigated mass mortalities of koi, Cyprinus carpio Linnaeus, 1758, experienced in South Indian fish farms by virus isolation, electron microscopy, PCR detection, sequencing of capsid protein gene and transmission studies. Samples of moribund koi brought to the laboratory suffered continuous mortality exhibiting swimming abnormalities, intermittent surfacing and skin darkening. Irido-like virus was isolated from the infected fish in the indigenous snakehead kidney cell line (SNKD2a). Icosahedral virus particles of 100 to 120 nm were observed in the infected cell cultures, budding from the cell membrane. Virus transmission and pathogenicity studies revealed that horizontal transmission occurred associated with mortality. PCR analysis of infected fish and cell cultures confirmed the presence of Ranavirus capsid protein sequences. Sequence analysis of the major capsid protein gene showed an identity of 99.9% to that of largemouth bass virus isolated from North America. Detection and successful isolation of this viral agent becomes the first record of isolation of a virus resembling Santee-Cooper Ranavirus from a koi and from India. We propose the name koi ranavirus to this agent.

Ultrastructural morphogenesis of an amphibian iridovirus isolated from Chinese giant salamander (Andrias davidianus).

Haemorrhagic disease of Chinese giant salamanders (Andrias davidianus) (CGSs) is an emerging condition caused by an iridovirus of the genus Ranavirus within the family Iridoviridae. Several studies have described different biological properties of the virus, but some aspects of its replication cycle, including ultrastructural alterations, remain unknown. The aim of the present study was to describe the morphogenesis of Chinese giant salamander iridovirus (GSIV) in an epithelioma papulosum cyprinid (EPC) cell line at the ultrastructural level. Cells were infected with GSIV at a multiplicity of infection (MOI) of 10 and examined at 1, 2, 4, 6, 12, 24, 48, 72, 84 and 96 h post infection. GSIV entered EPC cells by endocytosis or fusion after adsorption to the cell membrane. Following uncoating, the viral cores translocated to the nucleus and the virus began to replicate. Different stages of virus self-assembly were observed in the slightly electron-lucent viromatrix near the cell nucleus. In the late phase of virus infection, most nucleocapsids were mature and formed a typical icosahedral shape and aggregated in pseudocrystalline array at the viromatrix or were budding at the plasma membrane. Virus infection was readily detected by electron microscopy before cytopathic effect appeared in cell culture. The EPC cell line represents a suitable in-vitro model for study of GSIV morphogenesis and characterization of the GSIV replication cycle.

Investigation of ornamental fish entering the EU for the presence of ranaviruses.

A survey was performed on ornamental fish imported into the EU to detect viral agents belonging to the genus Ranavirus. The objective was to gain knowledge of the potential for these systemic iridoviruses to gain entry into the EU via international trade in ornamental fish. A total of 208 pooled samples, representing 753 individual fish, were tested. The samples included 13 orders and 37 families, originating from different countries and continents. Tissues from fish that died during or just after transport were collected and examined by standard virological techniques in epithelioma papulosum cyprini cells, by transmission electron microscopy and by PCR for the detection of the major capsid protein and DNA polymerase gene sequences of ranaviruses. Virus was isolated from nine fish species but ranavirus was not identified in those samples. The results suggest that ranaviruses are not highly prevalent in ornamental fish imported into the EU.

Electron microscopic examination of the viromatrix of Rana grylio virus in a fish cell line.

Rana grylio virus (RGV), a Ranavirus belonging to the family Iridoviridae, assembles in the viromatrix which is a factory for viral genome replication and particle assembly. Ultrastructural studies of the viromatrix will clarify the pathway of assembly. The viromatrix and quantitative changes in RGV infected epithelipma papulosum cyprini (EPC) cells, one of fish cell lines, were studied by electron microscopy. It was shown that viromatrices were adjacent to the nucleus, and the electron density was lower than that of the surrounding cytoplasm. The viromatrix contained virus particles with different forms, electron-dense materials and amorphous structures which included tubules and membranous materials. Tubules were often observed in direct continuity with empty capsids. Several bundles of intermediate filaments were seen alongside the viromatrix and crystalline aggregates. Large clusters of mitochondria occurred in proximity to viromatrix. A total of 990 cells profiles were examined. The results showed that 394 cells contained viromatrix: 89.3% contained one, and 10.7% contained two to four viromatrices. The number of viromatrices increased gradually and reached a peak at 16 h p.i. The viromatrix area at 24 h p.i. increased up to 7.4 +/- 0.69 microm(2) which was three-times lower than that at 6 h p.i. The number of empty capsids within viromatrix was generally more than that of "full" particles at different time points, and there was a strong positive correlation between them.

Inhibition of reporter gene and Iridovirus-tiger frog virus in fish cell by RNA interference.

We describe the specific silencing of reporter gene lacZ in FHM cells (muscle cells of fathead minnow, a fish cell line) by either expressing small hairpin RNAs (shRNAs) from plasmids or transfecting small interfering RNAs (siRNAs) transcribed in vitro. Two types of dsRNAs could inhibit reporter gene expression, and siRNAs were more effective, while both of them worked very well in HeLa cells. siRNAs were tested for silencing expression of the major capsid protein (MCP) encoded by tiger frog virus (TFV), an iridovirus causing severe disease in fish. siRNAs targeting mcp gene effectively inhibited TFV replication in fish cells as demonstrated by reduced mcp RNA level, postponed emergence of cytopathogenic effect, as well as reduced TFV titer and particles in cells. The results suggest that the siRNA method suppressed TFV efficiently in fish cells, providing a potential approach to the therapy of aquaculture viral diseases.

Characterization of a novel ranavirus isolated from grouper Epinephelus tauvina.

A large icosahedral virus was isolated from diseased grouper Epinephelus tauvina. The virus grew well in several cultured fish cell lines, with stable and high infectivity after serial passages in grouper cell line (GP). The virus was sensitive to both acid and heat treatments. Virus replication was inhibited by 5-iodo-2-deoxyuridine (IUDR), indicative of a DNA-containing genome. The virus infectivity was reduced with ether treatment, suggesting that the virus was lipid-enveloped. Electron micrographs showed abundant cytoplasmic icosahedral virons in the virus-infected GP cells. The size of the intracellular nucleocapsid was 154 nm between the opposite sides, or 176 nm between the opposite vertices with an inner electron-dense core of 93 nm. Virus particles were released through budding from plasma membranes with a size of 200 nm in diameter. SDS-PAGE of purified virus revealed 20 structural protein bands and a major capsid protein (MCP) of 49 kDa. A DNA fragment of approximately 500 nucleotides was successfully amplified by polymerase chain reaction (PCR) using the primers from conserved regions of the MCP gene of frog virus 3 (FV3), the type species of Ranavirus. Subsequent multiple alignment and phylogenetic analysis showed that the newly isolated grouper virus was closely related to largemouth bass virus (LMBV), FV3 and Regina ranavirus (RRV). Our data suggests that the virus isolate is a novel member of genus Ranavirus, family Iridoviridae. We tentatively name the virus as Singapore grouper iridovirus (SGIV). SGIV was able to cause serious systemic disease capable of killing 96% of grouper fry.

First identification of a ranavirus from green pythons (Chondropython viridis).

Ten juvenile green pythons (Chondropython viridis) died or were euthanized shortly after having been illegally imported into Australia from Indonesia in 1998. Histologic examination of two of the three snakes that died revealed moderately severe chronic ulceration of the nasal mucosa and focal or periacinar degeneration and necrosis of the liver. In addition there was severe necrotizing inflammation of the pharyngeal submucosa accompanied by numerous macrophages, heterophils, and edema. An iridovirus was isolated in culture from several tissues and characterized by immunohistochemistry, electron microscopy, enzyme-linked immunosorbent Assay, polyacrylamide gel electrophoresis, polymerase chain reaction and sequence analysis, restriction endonuclease digestion, and DNA hybridization. This is the first report of a systemic ranavirus infection in any species of snake and is a new member of the genus, Ranavirus.

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

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

Comparative studies of piscine and amphibian iridoviruses.

A total of 30 iridoviruses collected from Australia, South-East Asia, North America, South America and Europe were characterised. With the exception of the South-East Asian iridoviruses all viruses were found to belong to the genus Ranavirus. All viruses, except those originating from South-East Asia, cross-reacted with antisera against epizootic haematopoietic necrosis virus (EHNV). Viruses or virus-infected cells were examined using electron microscopy, SDS PAGE, restriction endonuclease (RE) digestion, DNA hybridisation, and DNA sequencing. Data from RE digestion of genomic DNA, and from the sequencing of PCR products indicated that the viruses generally grouped according to their geographic and taxonomic (i.e. amphibian or fish) origin. The one exception to this was the viruses from the United Kingdom that grouped with the North American ranaviruses. The differences between specified genomic regions were small. To assess the validity of the differences in sequence homology, similar studies were performed with different isolates from two viruses (EHNV and Guatopo virus (GV), collected from different animals at different locations and time). The sequence data showed complete homology for the isolates for any one virus over the 200 and 586 bp regions examined. Collectively, the data showed that the coding region for the major coat protein (MCP) is stable for any one species (e.g. EHNV).

Giant toads Bufo marinus in Australia and Venezuela have antibodies against 'ranaviruses'.

A serological survey was conducted for antibodies against 'ranaviruses' in the giant toad Bufo marinus in Australia and Venezuela. Sera containing antibodies against 'ranaviruses' were found in both countries. In Australia positive antibodies were identified in populations throughout most of the known range of B. marinus. Results were confirmed by immunofluorescence and immunoelectron microscopy where a characteristic staining pattern of 'ranaviruses' in infected cells was observed. Whilst a 'ranavirus(es)' has been isolated from populations of B. marinus in Venezuela, no virus has been isolated from Australian B. marinus populations. The significance of 'ranavirus' sero-positive B. marinus in Australia is discussed.

Isolation and characterization of iridoviruses from the giant toad Bufo marinus in Venezuela.

In this communication we describe for the first time the isolation of 7 iridoviruses from the toad Bufo marinus and an unknown species of frog Leptodactylus in Venezuela, South America. The viruses are icosahedral with electron-dense cores, each of which is surrounded by an inner membrane, capsid and a cell-derived envelope. The virus(es) have an average vertex to vertex diameter of 160 nm and replicate in the cytoplasm of a range of cell lines. Within the cytoplasm of infected cells, rarefied areas could be observed; structures lacked cellular organelles and contained complete, empty and developing viruses. Results from antigen-capture enzyme-linked immunosorbent assays (ELISA) with polyclonal antibody raised against epizootic haematopoietic necrosis virus (EHNV) indicated cross-reactivity between these isolates, Bohle iridovirus (BIV) and frog virus 3 (FV3). Comparison of polypeptide and genomic profiles indicated that the Venezuelan viruses shared many polypeptides of equivalent molecular weight with type species FV3. There were, however, differences between the group of Venezuelan viruses and FV3 and BIV. The viruses belongs to the family Iridoviridae and the genus Ranavirus.