Detection of shrimp hemocyte iridescent virus by recombinase polymerase amplification assay.

Shrimp hemocyte iridescent virus (SHIV), which was first identified in white leg shrimp (Litopenaeus vannamei) in China in 2014, can cause extensive shrimp mortality and major economic losses in the shrimp farming industry in China. In this study, a novel real-time isothermal recombinase polymerase amplification (RPA) assay was developed using a TwistAmp exo kit for SHIV detection. First, five primers and a probe were designed for the major capsid protein gene (GenBank: KY681039.1) according to the TwistDx manual; next, the optimal primers were selected by a comparison experiment. The primers and probe were specific for SHIV and did not react with shrimp white spot syndrome virus (WSSV), shrimp infectious hypodermal and hematopoietic necrosis virus (IHHNV), shrimp enterocytozoon hepatopenaei (EHP), and macrobrachium rosenbergii nodavirus (MrNV) samples, as well as pathogens of acute hepatopancreatic necrosis disease (AHPND). The RPA assay reached a detection limit of 11 copies per reaction according to probit regression analysis. In addition, RPA assay detected the positive plasmid samples at concentration of 1000 copies/µL within 16.04 ±â€¯0.72 min at a single low operation temperature (39 °C). The results proved that the proposed RPA method was an accurate, sensitive, affordable, and rapid detection tool that can be suitably applied for the diagnosis of SHIV in field conditions and in resource-poor settings.

In-depth proteomic profiling of the Singapore grouper iridovirus virion.

Singapore grouper iridovirus (SGIV) is a lethal grouper virus containing 162 predicted ORFs. Previous proteomic studies led to identification of 73 SGIV structural proteins. Here, SDS-assisted tube-gel digestion and DOC-assisted in-solution digestion coupled with LC-ESI-MS/MS were applied to further profile the SGIV structural proteome. We identified a total of 90 SGIV structural proteins including 24 newly reported proteins. Additionally, several PTMs were identified, including 26 N-terminal acetylated proteins, three phosphorylated proteins, and one myristoylated protein. Importantly, 47 of the proteins that were identified are predicted to contain conserved domains. Our work greatly expands the repertoire of the SGIV structural proteome and provides more insight into the biology of SGIV.

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

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

Development of a loop-mediated isothermal amplification assay for rapid detection of iridovirus in the Chinese giant salamander.

The Chinese giant salamander (Andrias davidianus) iridovirus (GSIV) is an emerging infectious pathogen responsible for severe hemorrhagic disease and high mortality in cultured Chinese giant salamanders. A loop-mediated isothermal amplification (LAMP) assay based on the major caspid protein (MCP) gene has been developed to detect this virus. Primer pairs for the LAMP assay were designed based on the GSIV MCP gene sequence. Amplification results indicate that under optimized conditions the LAMP assay has the ability to specifically detect the virus in both diseased animals and infected epithelioma papilloma cyprinid (EPC) cells. The assay was shown to be 10-fold more sensitive than nested PCR and was able to detect concentrations of 10(-9) (approximately 0.01 pg/µL). The LAMP assay is relatively easy to perform in situ and the amplification products can be observed directly under UV light or via staining with SYBR Green I. The LAMP assay is also rapid and cost-effective. This study establishes the use of a LAMP assay for rapid detection of GSIV, which is a novel and important tool for the diagnosis of GSIV infection in laboratory or farmed Chinese giant salamanders.

Mass-mortality in green striped tree dragons (Japalura splendida) associated with multiple viral infections.

In spring 2011, high mortality in association with skin lesions, systemic haemorrhages and necrosis occurred in a group of green striped tree dragons (Japalura splendida) which were imported from southwestern China via Florida to Germany. Infections with various endoparasites were diagnosed in coprological examinations. Different antiparasitic and antibiotic treatments over a period of three months did not reduce the mortality rate. The remaining animals were therefore euthanased and submitted for additional testing. Predominant findings in pathological examination were granulomatous and necrotising inflammation of the skin, vacuolar tubulonephrosis of the distal renal tubules, hyperaemia and liver necrosis. Eosinophilic intranuclear and basophilic intracytoplasmic inclusion bodies were detected in the liver. Virological testing (PCR and virus isolation methods) demonstrated the presence of ranavirus, adenovirus and invertebrate iridovirus.

Genetic analysis of fish iridoviruses isolated in Taiwan during 2001-2009.

To investigate the genetic relationships between field strains of iridoviruses gathered from various fish species in Taiwan, viruses that were collected from 2001 to 2009 were analyzed. Open reading frames encoding the viral major capsid protein (MCP) and adenosine triphosphatase (ATPase) were sequenced for phylogenetic analysis. Our results indicated that iridoviruses from Taiwan aquaculture fishes could be classified into two groups: prior to 2005, the viruses were closely related to members of the genus Ranavirus; and after 2005, they were similar to members of the genus Megalocytivirus. Based on the analysis of MCP amino acid sequences, virus isolates were divided into 4 major genotypes that were related to ISKNV, RSIV, FLIV, and GIV, respectively. Pairwise comparisons of MCP genes showed that the ranavirus was an epidemic pathogen for economically important species in the major production regions and cultured marine fish, while the megalocytivirus isolates were sensitive to host range. In addition, the distribution of synonymous and non-synonymous changes in the MCP gene revealed that the iridoviruses were evolving slowly, and most of the variations were synonymous mutations. The Ka/Ks values were lower than one, and hence, the viruses were under negative selection.

Complete genome sequence analysis of an iridovirus isolated from the orange-spotted grouper, Epinephelus coioides.

Orange-spotted grouper iridovirus (OSGIV) was the causative agent of serious systemic diseases with high mortality in the cultured orange-spotted grouper, Epinephelus coioides. Here we report the complete genome sequence of OSGIV. The OSGIV genome consists of 112,636 bp with a G+C content of 54%. 121 putative open reading frames (ORF) were identified with coding capacities for polypeptides varying from 40 to 1168 amino acids. The majority of OSGIV shared homologies to other iridovirus genes. Phylogenetic analysis of the major capsid protein, ATPase, cytosine DNA methyl transferase and DNA polymerase indicated that OSGIV was closely related to infectious spleen and kidney necrosis virus (ISKNV) and rock bream iridovirus (RBIV), but differed from lymphocytisvirus and ranavirus. The determination of the genome of OSGIV will facilitate a better understanding of the molecular mechanism underlying the pathogenesis of the OSGIV and may provide useful information to develop diagnosis method and strategies to control outbreak of OSGIV.

Iridovirus disease in two ornamental tropical freshwater fishes: African lampeye and dwarf gourami.

Many species of ornamental freshwater fishes are imported into Japan from all over the world. We found African lampeye Aplocheilichthys normani and dwarf gourami Colisa lalia suffering from an iridovirus infection just after being imported by tropical fish wholesalers from Singapore. African lampeye were cultured on the Indonesian Island of Sumatra and dwarf gourami were cultured in Malaysia before export. Diseased fishes displayed distinct histopathological signs of iridovirus infection: systemic appearance of inclusion body-bearing cells, and necrosis of splenocytes and hematopoietic cells. Electron microscopy revealed viral particles (African lampeye:180 to 200 nm in edge to edge diameter; dwarf gourami: 140 to 150 nm in diameter) in an inclusion body within the cytoplasm of inclusion body-bearing cells as well as in the cytoplasm of necrotized cells. Experimental infection with an iridovirus isolate from African lampeye (ALIV) revealed pathogenicity of ALIV to African lampeye and pearl gourami Trichogaster leeri. Polymerase chain reaction (PCR) products from ALIV and an iridovirus isolate from dwarf gourami (DGIV) using iridovirus-specific primers were indistinguishable. The nucleotide sequence of PCR products derived from ALIV (696 base pairs) and DGIV (701 base pairs) had 95.3% identity. These results indicate that ALIV and DGIV have a single origin.

Sequence analysis of the complete genome of an iridovirus isolated from the tiger frog.

We have isolated a tiger frog virus (TFV) from diseased tiger frogs, Rana tigrina rugulosa. The genome was a linear double-stranded DNA of 105,057 basepairs in length with a base composition of 55.01% G+C. About 105 open reading frames were identified with coding capacities for polypeptides ranging from 40 to 1294 amino acids. Computer-assisted analyses of the deduced amino acid sequences revealed that 39 of 105 putative gene products showed significant homology to functionally characterized proteins of other species in the GenBank/EMBL/DDBJ databases. These proteins included enzymes and structural proteins involved in virus replication, transcription, modification, and virus--host interaction. The deduced amino acid sequences of TFV gene products showed more than 90% identity to FV3, but a low degree of similarity among TFV, ISKNV, and LCDV-1. The results from this study indicated that TFV may belong to the genus Ranavirus of the family Iridoviridae.

Characterization of an iridescent virus isolated from Gryllus bimaculatus (Orthoptera: Gryllidae).

We have isolated an iridescent virus from commercially produced colonies of Gryllus bimaculatus in Germany, which showed apparent mortality. Transmission electron microscopy studies on adult cricket specimens revealed the paracrystalline assembly of icosahedral virus particles in the cytoplasm of hypertrophied abdominal fat body cells. The infecting agent could be cultivated in the lepidopteran cell line sf-9, where it caused cytopathogenic effects such as cell hypertrophy, cytoplasmic vacuolization, and cell death within 8 days postinfection. Infection titers of the first virus passage reached 10(7.5) TCID(50)/ml. Negatively stained virus particles (n = 100) had dimensions of 172 +/- 6 nm (apex to apex) and 148 +/- 5 nm (side to side). SDS-polyacrylamide gel electrophoresis of virus proteins showed more than 20 distinct polypeptides with a major species of approximately 50 kDa. Analysis of the restriction fragment length profiles from digestion of purified viral DNA with the endonucleases EcoRI, BamHI, and HindIII showed marked differences from the profiles of iridoviruses of lower vertebrates (genus Ranavirus), e.g., Rana esculenta Iridovirus and Frog virus 3. Restriction enzyme digests with the endonucleases MspI and HpaII indicated the lack of methylation of viral DNA. Polymerase chain reaction led to the amplification of a 420-bp gene fragment with 97% sequence homology to the major capsid protein gene of Chilo iridescent virus. These data indicate that this new isolate, which we propose to be termed Gryllus bimaculatus iridescent virus, belongs to the genus Iridovirus of the family Iridoviridae.

Characterization of an iridovirus from the cultured pig frog Rana grylio with lethal syndrome.

Three virus isolates, RGV-9506, RGV-9807 and RGV-9808, were obtained from cultured pig frogs Rana grylio undergoing lethal infections. Previously, the first isolate, RGV-9506, was shown to be an iridovirus based on ultrastructural and morphological studies. In the present study, the original isolate, along with 2 recent ones, were more extensively characterized by experimental infection studies, histopathology, electron microscopy, serological reactivity, gel electrophoresis of viral polypeptides and DNA restriction fragments, PCR amplification, and nucleic acid sequence analysis of the major capsid protein (MCP) gene. The 3 isolates were shown to be identical to each other, and very similar to FV3, the type species of the genus Ranavirus (family Iridoviridae). These results suggest that RGV should be considered a strain of FV3, and indicate that FV3-like iridoviruses are capable of causing widespread, severe disease among cultured frogs.

Pathology, isolation, and preliminary molecular characterization of a novel iridovirus from tiger salamanders in Saskatchewan.

All iridovirus was confirmed to be the cause of an epizootic in larval and adult tiger salamanders (Ambystoma tigrinum diaboli) from four separate ponds in southern Saskatchewan (Canada) during the summer of 1997. This organism also is suspected, based on electron microscopic findings, to be the cause of mortality of larval tiger salamanders in a pond over 200 km to the north during the same year. Salamanders developed a generalized viremia which resulted in various lesions including: necrotizing, vesicular and ulcerative dermatitis; gastrointestinal ulceration; and necrosis of hepatic, splenic, renal, lymphoid, and hematopoietic tissues. In cells associated with these lesions, large lightly basophilic cytoplasmic inclusions and vacuolated nuclei with marginated chromatin were consistently found. Virus was isolated from tissue homogenates of infected salamanders following inoculation of epithelioma papilloma cyprini (EPC) cells. The virus, provisionally designated Regina ranavirus (RRV), was initially identified as an iridovirus by electron microscopy. Subsequent molecular characterization, including partial sequence analysis of the major capsid protein (MCP) gene, confirmed this assignment and established that RRV was a ranavirus distinct from frog virus 3 (FV3) and other members of the genus Ranavirus. Intraperitoneal inoculation of 5 x 10(6.23) TCID50 of the field isolate caused mortality in inoculated salamanders at 13 days post infection. Field, clinical, and molecular studies jointly suggest that the etiological agent of recent salamander mortalities is a highly infectious novel ranavirus.

Pathological and microbiological findings from incidents of unusual mortality of the common frog (Rana temporaria).

In 1992 we began an investigation into incidents of unusual and mass mortalities of the common frog (Rana temporaria) in Britain which were being reported unsolicited to us in increasing numbers by members of the public. Investigations conducted at ten sites of unusual mortality resulted in two main disease syndromes being found: one characterized by skin ulceration and one characterized by systemic haemorrhages. However, frogs also were found with lesions common to both of these syndromes and microscopic skin lesions common to both syndromes were seen. The bacterium Aeromonas hydrophila, which has been described previously as causing similar lesions, was isolated significantly more frequently from haemorrhagic frogs than from those with skin ulceration only. However, as many of the latter were euthanased, this may have been due to differences in post mortem bacterial invasion. An iridovirus-like particle has been identified on electron microscopical examination of skin lesions from frogs with each syndrome and iridovirus-like inclusions have been detected in the livers of frogs with systemic haemorrhages. Also, an adenovirus-like particle has been cultured from one haemorrhagic frog. A poxvirus-like particle described previously from diseased frogs has now been found also in control animals and has been identified as a melanosome. Both the prevalence of the iridovirus-like particle and its association with lesions indicate that it may be implicated in the aetiology of the disease syndromes observed. Specifically, we hypothesize that primary iridovirus infection, with or without secondary infection with opportunistic pathogens such as A. hydrophila, may cause natural outbreaks of 'red-leg', a disease considered previously to be due to bacterial infection only.

Comparative studies of iridoviruses: further support for a new classification.

Changes in the classification of invertebrate iridoviruses (IVs) (Iridoviridae) have recently been proposed (Williams and Cory, 1994). The previous system of naming isolates according to the host and sequence of discovery (IV type 1, IV2, IV3, etc.) is not adequate for the purposes of taxonomy, since iridovirus isolates may infect many species, including hosts from diverse invertebrate orders. The new system of invertebrate iridovirus nomenclature, as with several other virus families, is based on geographical origin. Proposals have been made, based on DNA hybridization and other characteristics, by which invertebrate iridovirus isolates can be assigned to one of four recognized complexes, or considered as candidates for alternative assignations. This study reports comparative data on the DNA of 14 invertebrate iridovirus isolates used in the Williams and Cory study plus the two type vertebrate iridoviruses, frog virus 3 and flounder lymphocystis disease virus. DNA studies support the validity of assigning several isolates a common name and of grouping the known isolates into four complexes. The detection of such complexes is in broad agreement with previous serological studies. A previously undescribed isolate (San Miguel IV) obtained from the lepidopteran pest Anticarsia gemmatalis (Lep.: Noctuidae) has been initially characterized following the procedures recommended by Williams and Cory. DNA hybridization and Southern blot analysis identified this isolate as a new member of the Polyiridovirus complex. The San Miguel IV MSP gene was identified and a central fragment of ca. 719 bp was recovered by PCR amplification. The restriction endonuclease profiles (5 enzymes) of this isolate were distinct from others previously described.