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Ranaviruses are globally emerging pathogens negatively impacting wild and cultured fish, amphibians, and reptiles. Although conventional and diagnostic real-time PCR (qPCR) assays have been developed to detect ranaviruses, these assays often have not been tested against the known diversity of ranaviruses. Here we report the development and partial validation of a TaqMan real-time qPCR assay. The primers and TaqMan probe targeted a conserved region of the major capsid protein (MCP) gene. A series of experiments using a 10-fold dilution series of Frog virus 3 (FV3) MCP plasmid DNA revealed linearity over a range of 7 orders of magnitude (107-101), a mean correlation coefficient (R2) of >0.99, and a mean efficiency of 96%. The coefficient of variation of intra- and inter-assay variability ranged from Cyprinivirus. DNA from fish tissue homogenates previously determined to be positive or negative for the ranavirus Epizootic hematopoietic necrosis virus by virus isolation demonstrated a diagnostic sensitivity of 95% and a diagnostic specificity of 100%. The reported qPCR assay provides an improved expedient diagnostic tool and can be used to elucidate important aspects of ranaviral pathogenesis and epidemiology in clinically and sublinically affected fish, amphibians, and reptiles.

Ranavirus is the second most common infectious cause of amphibian mortality. These viruses affect caudates, an order in which information regarding Ranavirus pathogenesis is scarce. In the Netherlands, two strains (CMTV-NL I and III) were suspected to possess distinct pathogenicity based on field data. To investigate susceptibility and disease progression in urodeles and determine differences in pathogenicity between strains, 45 adult smooth newts (Lissotriton vulgaris) were challenged via bath exposure with these ranaviruses and their detection in organs and feces followed over time by PCR, immunohistochemistry and in situ hybridization. Ranavirus was first detected at 3 days post infection (p.i.) in the oral cavity and upper respiratory mucosa. At 6 days p.i, virus was found in connective tissues and vasculature of the gastrointestinal tract. Finally, from 9 days p.i onwards there was widespread Ranavirus disease in various organs including skin, kidneys and gonads. Higher pathogenicity of the CMTV-NL I strain was confirmed by higher correlation coefficient of experimental group and mortality of challenged animals. Ranavirus-exposed smooth newts shed virus in feces intermittently and infection was seen in the absence of lesions or clinical signs, indicating that this species can harbor subclinical infections and potentially serve as disease reservoirs.

Diseases are an important cause of losses and decreased production rates in freshwater eel farming, and have been suggested to play a contributory role in the worldwide decline in wild freshwater eel stocks. Three commonly detected pathogenic viruses of European eel Anguilla anguilla are the aquabirnavirus eel virus European (EVE), the rhabdovirus eel virus European X (EVEX), and the alloherpesvirus anguillid herpesvirus 1 (AngHV1). In general, all 3 viruses cause a nonspecific haemorrhagic disease with increased mortality rates. This review provides an overview of the current knowledge on the aetiology, prevalence, clinical signs and gross pathology of these 3 viruses. Reported experimental infections showed the temperature dependency and potential pathogenicity of these viruses for eels and other fish species. In addition to the published literature, an overview of the isolation of pathogenic viruses from wild and farmed A. anguilla in the Netherlands during the past 2 decades is given. A total of 249 wild A. anguilla, 39 batches of glass eels intended for farming purposes, and 239 batches of farmed European eels were necropsied and examined virologically. AngHV1 was isolated from wild A. anguilla yellow and silver eels from the Netherlands from 1998 until the present, while EVEX was only found sporadically, and EVE was never isolated. In farmed A. anguilla AngHV1 was also the most commonly isolated virus, followed by EVE and EVEX.

The order Herpesvirales encompasses viruses that share structural, genetic, and biological properties. However, members of this order infect hosts ranging from molluscs to humans. It is currently divided into three phylogenetically related families. The Alloherpesviridae family contains viruses infecting fish and amphibians. There are 12 alloherpesviruses described to date, 10 of which infect fish. Over the last decade, cyprinid herpesvirus 3 (CyHV-3) infecting common and koi carp has emerged as the archetype of fish alloherpesviruses. Since its first description in the late 1990s, this virus has induced important economic losses in common and koi carp worldwide. It has also had negative environmental implications by affecting wild carp populations. These negative impacts and the importance of the host species have stimulated studies aimed at developing diagnostic and prophylactic tools. Unexpectedly, the data generated by these applied studies have stimulated interest in CyHV-3 as a model for fundamental research. This review intends to provide a complete overview of the knowledge currently available on CyHV-3.

International audience; Eel virus European X (EVEX) was first isolated from diseased European eel Anguilla anguilla in Japan at the end of seventies. The virus was tentatively classified into the Rhabdoviridae family on the basis of morphology and serological cross reactivity. This family of viruses is organized into six genera and currently comprises approximately 200 members, many of which are still unassigned because of the lack of molecular data. This work presents the morphological, biochemical and genetic characterizations of EVEX, and proposes a taxonomic classification for this virus. We provide its complete genome sequence, plus a comprehensive sequence comparison between isolates from different geographical origins. The genome encodes the five classical structural proteins plus an overlapping open reading frame in the phosphoprotein gene, coding for a putative C protein. Phylogenic relationship with other rhabdoviruses indicates that EVEX is most closely related to the Vesiculovirus genus and shares the highest identity with trout rhabdovirus 903/87.

Viral interleukin 10 (IL-10) like open reading frames have been identified in several pox- and herpesviruses, including the fish herpesviruses Anguillid herpesvirus 1 (AngHV-1) and Cyprinid herpesvirus 3 (CyHV-3). European eel (Anguilla anguilla) IL-10 was sequenced, in order to compare European eel and common carp (Cyprinus carpio) IL-10 with their alloherpesviral counterparts. Homology between the virus and host IL-10 amino acid sequences is low, which is confirmed by phylogenetic analysis. However, the three dimensional structures of the fish and alloherpesviral IL-10 proteins as predicted by modeling are highly similar to human IL-10. Closely related AngHV-1 and CyHV-3 are expected to have obtained their viral IL-10 genes independently in the course of coexistence with their respective hosts. The presence and structural conservation of these alloherpesviral IL-10 genes suggest that they might play an important role in the evolution of pathogenesis.

Eel virus European X (EVEX) is one of the most common pathogenic viruses in farmed and wild European eel (Anguilla anguilla) in the Netherlands. The virus causes a hemorrhagic disease resulting in increased mortality rates. Cell culture and antibody-based detection of EVEX are laborious and time consuming. Therefore, a two-step real-time reverse transcriptase (RT-)PCR assay was developed for rapid detection of EVEX. Primers and probe for the assay were designed based on a sequence of the RNA polymerase or L gene of EVEX. The real-time RT-PCR assay was validated both for use with SYBR Green chemistry and for use with a TaqMan probe. The assay is sensitive, specific, repeatable, efficient and has a high r2-value. The real-time RT-PCR assay was further evaluated by testing field samples of European eels from the Netherlands, which were positive or negative for EVEX by virus isolation followed by an indirect fluorescent antibody test. The real-time RT-PCR assay allows rapid, sensitive and specific laboratory detection of EVEX in RNA extracts from 10% eel organ suspensions and cell cultures with cytopathic effects, and is a valuable contribution to the diagnosis of viral diseases of eel.

Eel herpesvirus or anguillid herpesvirus 1 (AngHV1) frequently causes disease in freshwater eels. The complete genome sequence of AngHV1 and its taxonomic position within the family Alloherpesviridae were determined. Shotgun sequencing revealed a 249 kbp genome including an 11 kbp terminal direct repeat that contains 7 of the 136 predicted protein-coding open reading frames. Twelve of these genes are conserved among other members of the family Alloherpesviridae and another 28 genes have clear homologues in cyprinid herpesvirus 3. Phylogenetic analyses based on amino acid sequences of five conserved genes, including the ATPase subunit of the terminase, confirm the position of AngHV1 within the family Alloherpesviridae, where it is most closely related to the cyprinid herpesviruses. Our analyses support a recent proposal to subdivide the family Alloherpesviridae into two sister clades, one containing AngHV1 and the cyprinid herpesviruses and the other containing Ictalurid herpesvirus 1 and the ranid herpesviruses.

Background: Whereas temporal gene expression in mammalian herpesviruses has been studied extensively, little is known about gene expression in fish herpesviruses. Here we report a genome-wide transcription analysis of a fish herpesvirus, anguillid herpesvirus 1, in cell culture, studied during the first 6 hours of infection using reverse transcription quantitative PCR.\ud \ud Results: Four immediate-early genes – open reading frames 1, 6A, 127 and 131 – were identified on the basis of expression in the presence of a protein synthesis inhibitor and unique expression profiles during infection in the absence of inhibitor. All of these genes are located within or near the terminal direct repeats. The remaining 122 open reading frames were clustered into groups on the basis of transcription profiles during infection. Expression of these genes was also studied in the presence of a viral DNA polymerase inhibitor, enabling classification into early, early-late and late genes. In general, clustering by expression profile and classification by inhibitor studies corresponded well. Most early genes encode enzymes and proteins involved in DNA replication, most late genes encode structural proteins, and early-late genes encode non-structural as well as structural proteins.\ud \ud Conclusions: Overall, anguillid herpesvirus 1 gene expression was shown to be regulated in a temporal fashion, comparable to that of mammalian herpesviruses.