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Carp edema virus disease (CEVD) is a severe viral illness that causes substantial economic losses in wild and farmed common carp and koi. It is caused by carp edema virus (CEV), a member of the Poxviridae family , whose genetic diversity and genome evolution are poorly understood. Based on a genomic fragment of the 4a gene, two genogroups, genogroup I (gI) and genogroup II (gII), have been identified in samples of different origins. By analysing a series of recent samples, we highlight here a new genomic region of interest that varies by substitutions, indels and putative recombinations. In the Japanese reference sequence, this region encodes an ORF, cds46, whose function is unknown despite weak homologies with genes of some members of the Iridoviridae . Surprisingly, AlphaFold protein structure prediction analyses link cds46-encoded ORF with cellular endonucleases, providing insights into its possible origin. The ORF is absent in all gI haplotypes and in some gII haplotypes. Apart from the absence of cds46, gI haplotypes show an insertion of 121 bp with no homology to any viral sequence. When present, cds46 showed two groups of alleles differentiated by substitutions. The analysis of the cds46 locus showed that some samples from fish batches contained mixes of different haplotypes, irrespective of their origin (i.e. France, Japan or Israel). In a 2023 sample, we also found a virus carrying a gII-like atypical 4a allele first identified in France in 2015, indicating the limited but persistent spread of this virus in the country. The cds46 locus is a new target that may be useful for identifying and tracking CEV haplotypes.

Variants of perch rhabdovirus (PRV) circulate across European percid farms via the fish trade. To trace their circulation, they are usually isolated by cell culture and subsequently identified genetically by sequencing partial or complete genes. Here, a newly developed nested PCR-based method was used to amplify and sequence the complete N and P genes directly from clinical samples obtained during an outbreak on a farm as well as from four batches of fish sampled from two other farms in another country. In an attempt to trace the origin of the five detected viruses, their N and P sequences were concatenated and compared with related viruses. One virus found in pike-perch was highly related to a virus isolated in 2016 in Belgium. Two other viruses detected on a single farm were distinct from one another, with one being almost identical to another virus isolated in 2016 in Belgium and the other being more closely related to a subgroup with different origins, France and Belgium. Two other viruses found in perch from a third farm were identical and were more related to a subgroup of viruses isolated in France. Identifying variants by a direct PCR approach will help to prevent further dissemination in farms., (© 2022 John Wiley & Sons Ltd.)

The koi sleepy disease of carp caused by the carp oedema virus (CEV) was observed on farms and in ponds in France since the 2010s. Samples of CEV collected in France over a period of eight years were characterized at the molecular level by sequencing the partial p4a gene. All the sequences, except one, fell into two well-defined genogroups. Sequences obtained from CEV detected in common carp generally clustered in genogroup I and sequences from CEV detected in the koi were assigned to genogroup II. A particular sample was different to the others and represented a putative new genogroup possibly arose from a recombination event between a genogroup II sequence and one from an unknown genogroup. Compared with sequences from CEV of other countries, most of the French sequences exhibited high degree of DNA identities with those published previously, indicating identical sources of viruses. The sequence diversity suggests multiple introductions of the viruses in France. Among the French sequences, two genogroup-specific molecular markers were identified. One was an insertion/deletion identified within a microsatellite and other was a group of single nucleotide polymorphisms. CEV seems to generate genetic diversity via diverse mechanisms: substitutions, indels and recombination events., (© 2021 John Wiley & Sons Ltd.)

A group of pathogenic nucleocytoplasmic large DNA viruses (NCLDVs) related to the Mimiviridae family infect farmed sturgeons across Europe, causing mild-to-severe losses. One of these viruses, Acipenser iridovirus-European (AcIV-E), was identified in six sturgeon species. During the 2018-2019 period, nine sick Siberian (A. baerii) and Russian (A. gueldenstaedtii) sturgeons were sampled in Ukrainian farms and tested for the presence of AcIV-E using real-time PCR. The presence of AcIV-E was confirmed in some samples. High-resolution melting (HRM) assay and Sanger sequencing demonstrated the presence in three farms of two alleles of the major capsid protein (MCP) gene, called var1 and var2. Five samples carried both var1 and var2 at varying ratios, and the sixth sample was infected with only var1. These results constitute the first detection of AcIV-E in Ukraine and the first detection of a sample carrying only var1. The full-length sequences of the MCP genes confirmed the existence of two genetic lineages of AcIV-E, tentatively named V1 and V2, each displaying multiple substitutions in the MCP gene. Some of the MCP sequences showed a genetic relationship to both V1 and V2 lineages, depending on the fragment examined. Most likely, these sequences resulted from recombination events., (© 2020 John Wiley & Sons Ltd.)

Perhabdoviruses are a threat to some freshwater fish species raised in aquaculture farms in Europe. Although the genetic diversity of these viruses is suspected to be high, the classification of isolates is still in its infancy, with just one full-length genome available and only partial sequences for a limited number of others. Here, we characterized a series of viruses isolated from percids in France from 1999 to 2009 by sequencing the nucleoprotein (N) gene. Four main clusters were distinguished, all related at varying levels of similarity to one of the two already-recognized species, namely Perch perhabdovirus and Sea trout perhabdovirus . Furthermore, we obtained the complete genome of five isolates, including one belonging to Sea trout rhabdovirus . The analysis of the complete L genes and the concatenated open reading frames confirmed the existence of four main genetic clusters, sharing 69 to 74% similarity. We propose the assignation of all these viral isolates into four species, including two new ones: Perch perhabdovirus 1 , Perch perhabdovirus 2 , Sea trout perhabdovirus 1 and Sea trout perhabdovirus 2 . In addition, we developed new primers to readily amplify specific portions of the N gene of any isolate of each species by conventional PCR. The presence of such genetically diverse viruses in France is likely due to divergent viral populations maintained in the wild and then introduced to experimental facilities or farms, as well as via trade between farms across the European continent. It is now urgent to improve the identification tools for this large group of viruses to prevent their unchecked dissemination.

Acipenser iridovirus-European (AcIV-E) is an important pathogen of sturgeons. Two variants differing by single-nucleotide polymorphisms (SNP) in the Major Capsid Protein gene have been described, but without any indication as to their prevalence in farms. To facilitate epidemiological studies, we developed a high-resolution melting (HRM) assay to distinguish between two alleles (var1 and var2) differing by five point substitutions. The HRM assay detected as little as 100 copies of plasmids harboring cloned sequences of var1 and var2, which have melting temperatures (Tm) differing by only 1 °C. The assay was specific of AcIV-E as demonstrated by the absence of signal when testing a related, yet distinct, virus as well as DNA from an AcIV-E-negative sturgeon sample. Experiments with mixtures of two distinct plasmids revealed abnormal melting curve patterns, which showed dips just before the main melting peaks. These dips in the curves were interpreted as the dissociation of heteroduplexes fortuitously created during the PCR step. Screening AciV-E-positive field samples of Russian sturgeons from three farms revealed the presence of var2, based on the Tm. However, for a few samples, the melting curves showed patterns typical of var2 as the dominant viral genome, mixed with another minor variant which proved to be var1. In conclusion, HRM is a simple method to screen for AcIV-E var1 and var2 and can be used on a large scale in Europe to trace these two variants which likely represent two genetic lineages., (Copyright © 2018 Elsevier B.V. All rights reserved.)

New genomic sequence data were acquired for the Acipenser iridovirus-European (AcIV-E), a virus whose complete genome and classification still remain to be elucidated. Here, we obtained the first full-length Major capsid protein (MCP) gene sequence for AcIV-E, as well as two additional open reading frames (ORFs) adjacent to the MCP gene. BLAST searches of the first ORF (α) resulted in no match to any gene or protein in the public databases. The other ORF (β) was identified as a subunit of a replication factor C (RFC), known to function as a clamp loader in eukaryotes, archae and some viruses. The presence of similar RFC genes was confirmed in two distinct, yet related, viruses, the white sturgeon iridovirus and a European variant of Namao virus. The existence of an RFC gene in AcIV-E suggests a genome size larger than that of other classifiable members of the family Iridoviridae along with a mode of replication involving an interaction between a clamp loader and a proliferating nuclear cell antigen. Sequencing and comparison of the full-length RFC gene from various sturgeon samples infected with AcIV-E revealed two distinct clusters of sequences within one particular sample in which the coexistence of two lineages had previously been predicted based on analysis of the partial MCP gene sequence. These genetic data provide further evidence of the circulation of at least two concurrent AcIV-E lineages, sometimes co-infecting cultured European sturgeon.

In 2016, a total of 5 massive mortality episodes each affecting hundreds of thousands of pike-perch Sander lucioperca larvae occurred at 2 sites in 2 Western European countries. For each episode, perhabdoviruses related to the perch rhabdovirus (PRV) were detected in samples, using either PCR or cell culture combined with PCR. The sequences of the glycoprotein (g), phosphoprotein (p) and nucleoprotein (n) genes of these samples demonstrated that 2 different genotypes were present at 1 site, each associated with 1 of the 3 episodes. At the other site, a single genotype was associated with the 2 outbreaks. Furthermore, this genotype was strictly identical to 1 genotype involved in the outbreaks of the first site, strongly suggesting a common origin for these 2 viruses. The common origin was confirmed a posteriori because some larvae introduced to both sites had exactly the same geographic origin in Eastern Europe. Taken together, the molecular and epidemiological data suggest that both horizontal and vertical transmission of 2 distinct strains of perhabdoviruses were involved in the various outbreaks affecting pike-perch.

Carp edema virus disease (CEVD), also known as koi sleepy disease, is caused by a poxvirus associated with outbreaks of clinical disease in koi and common carp Cyprinus carpio. Originally characterised in Japan in the 1970s, international trade in koi has led to the spread of CEV, although the first recognised outbreak of the disease outside of Japan was not reported until 1996 in the USA. In Europe, the disease was first recognised in 2009 and, as detection and diagnosis have improved, more EU member states have reported CEV associated with disease outbreaks. Although the structure of the CEV genome is not yet elucidated, molecular epidemiology studies have suggested distinct geographical populations of CEV infecting both koi and common carp. Detection and identification of cases of CEVD in common carp were unreliable using the original PCR primers. New primers for conventional and quantitative PCR (qPCR) have been designed that improve detection, and their sequences are provided in this paper. The qPCR primers have successfully detected CEV DNA in archive material from investigations of unexplained carp mortalities conducted >15 yr ago. Improvement in disease management and control is possible, and the principles of biosecurity, good health management and disease surveillance, applied to koi herpesvirus disease, can be equally applied to CEVD. However, further research studies are needed to fill the knowledge gaps in the disease pathogenesis and epidemiology that, currently, prevent an accurate assessment of the likely impact of CEVD on European koi and common carp aquaculture and on wild carp stocks.

Iridoviridae are known to cause disease in sturgeons in North America. Here, histological and molecular methods were used to screen for this family of virus in sturgeons from various European farms with low-to-high morbidity. Some histological samples revealed basophilic cells in the gill and labial epithelia, strongly suggesting the accumulation of iridovirus particles. Newly developed generic PCR tests targeting the major capsid protein (MCP) gene of sturgeon iridoviruses identified in North America, namely the white sturgeon iridovirus and the Namao virus (NV), produced positive signals in most samples from four sturgeon species: Russian (Acipenser gueldenstaedtii), Siberian (A. baerii), Adriatic (A. naccarii) and beluga (Huso huso). The sequences of the PCR products were generally highly similar one another, with nucleotide identities greater than 98%. They were also related to (74-88%), although distinct from, American sturgeon iridoviruses. These European viruses were thus considered variants of a single new virus, provisionally named Acipenser iridovirus-European (AcIV-E). Moreover, three samples infected with AcIV-E showed genetic heterogeneity, with the co-existence of two sequences differing by five nucleotides. One of our European samples carried a virus distinct from AcIV-E, but closely related to NV identified in Canada (95%). This study demonstrates the presence of two distinct sturgeon iridoviruses in Europe: a new genotype AcIV-E and an NV-related virus., (© 2016 John Wiley & Sons Ltd.)

Massive mortalities of Carassius auratus (L.) occurred in a farm in France during summer 2014. Fish presented anorexia, loss of scales and large amounts of mucus on the gills. Necrosis of the distal tip of the filament and the lamellae, combined with fusion of the lamellae, was observed, as well as necrosis in the hematopoietic organs and in the digestive tract. The histological examination led to hypothesize the implication of a virus in the mortality. The presence of cyprinid herpesvirus 2 (CyHV-2) in dead fish was demonstrated by amplification and sequencing of portions of the DNA polymerase and helicase genes, both sequences exhibiting 100% identity with CyHV-2 from Japan. In an attempt to find genetic markers of variation, two regions containing tandem repeats in the Japanese genome were amplified from a virus-positive sample from the present outbreak. A first region (mB) was fully identical to the Japanese isolate. However, the second region (mA) exhibited a range of deletions and substitutions compared to CyHV-2 from Japan. This is the first report of CyHV-2 in France in association with mortality of goldfish and the first identification of a molecular marker for its tracing., (© 2015 John Wiley & Sons Ltd.)

Viral nervous necrosis (VNN) is a severe neuropathological disease affecting a broad variety of finfish species worldwide. The causative agents of VNN are small viruses with a bi-segmented RNA genome known as betanodaviruses. At least four species with distinct but yet insufficiently characterized epidemiological features are recognized. The spread of VNN to an increasing number of host species, its wide geographic extent and its economical and ecological impacts justify the importance of collating as much molecular data as possible for tracing the origin of viral isolates and highlight the need for a freely accessible tool for epidemiological and molecular data sharing and consultation. For this purpose, we established a web-based specific database using the www.fishpathogens.eu platform, with the aim of collecting molecular and epidemiological information on VNN viruses, with relevance to their control, management and research studies., (© 2015 John Wiley & Sons Ltd.)

The detection of betanodavirus genomic components is a major issue for diagnostics and control of viral nervous necrosis (VNN), a devastating disease affecting fish worldwide. Despite a number of published molecular-based tests, most of them targeting the RNA2 molecule of the virus, diagnostics is still a challenge due to the high genetic diversity within this genus. In the present study, a new one-step real-time RT-PCR (rRT-PCR), targeting RNA1 of most genotypes of betanodaviruses, was proposed and validated. The test detected successfully various isolates of betanodavirus representatives of the four species RGNNV, SJNNV, TPNNV and BFNNV, either produced on cell culture or from clinical samples. It was specific as shown by the absence of signal on samples from healthy sea bass or from field samples of six other fish species without clinical signs of VNN. The assay detected reliably 50-100 copies of plasmids containing the targeted cloned RNA1 region, as well as an infectious dose of virus of 10(2.5)-10(2.85) TCID50/ml. A set of samples was tested by two different laboratories, with similar results, demonstrating the robustness of the test. This is the first one step generic rRT-PCR method for betanodaviruses. It is simple to perform and may be used for first intention diagnostics as well as for confirmation in case of doubtful results obtained with other published tests targeting RNA2.

A rhabdovirus was isolated in cell culture inoculated with tissue material from diseased grayling, Thymallus thymallus (L.), originating from a fish farm affected by a mortality episode in Poland. Diagnostics tests showed that the virus was not related to novirhabdoviruses known in Europe, nor to vesiculovirus-like species, except perch rhabdovirus (PRhV) with which it shared moderate serological relations. However, RT-PCR with PRhV probes gave negative results. To identify the virus, a random-priming sequence-independent single primer amplification was adopted. Surprisingly, two of the obtained sequences exhibited a high identity (>99%) with hirame rhabdovirus (HIRRV), a novirhabdovirus usually found in fish in marine Asiatic countries, for instance Japan, China and Korea. The full-length sequence of the phosphoprotein gene (P) demonstrated a higher identity of the present isolate with HIRRV from China compared with the Korean isolate. An identical viral sequence was also found in brown trout, Salmo trutta trutta L., affected by mortalities in a second farm in the same region, after a likely contamination from the grayling farm. To our knowledge, this is the first report of HIRRV in Europe, and in two hosts from fresh water that have not been described before as susceptible species., (© 2013 John Wiley & Sons Ltd.)

Viral nervous necrosis (VNN) is a serious viral disease affecting farmed sea bass (Dicentrarchus labrax). Only scarce molecular data are available on the disease-causing betanodavirus populations in Tunisia. Therefore, we carried out the first molecular survey of betanodaviruses in farmed sea bass and sea bream (Sparus aurata) along the Tunisian coasts. Among 81 samples from five farms, 20 tested positive with RT-PCR, not only in clinical cases but also in asymptomatic fish before and during outbreaks. Positive fish were found in all farms, except in one farm investigated in the south of Tunisia. Sequencing the fragments of both genomic components (RNA1 and RNA2) in 16 isolates revealed that the Tunisian viruses were related to the red-spotted grouper nervous necrosis virus (RGNNV) genotype. Furthermore, the newly sequenced isolates were generally highly related to one another suggesting a recent common ancestor. They also showed high identities with other isolates obtained from wild fishes in the Mediterranean, but were slightly more divergent from strains recently obtained from farmed fishes in the Mediterranean. The poor genetic diversity of the viral population along the Tunisian coasts is striking. One hypothesis is that it is the result of the maintenance of a homogenous genetic pool among infected wild fish, groupers for instance and subsequent dissemination to farmed fish over the seasons.

Cyprinid herpesvirus 3 (CyHV-3), the causative agent of koi herpesvirus disease, is a major threat for carp populations in many countries worldwide, including Indonesia. It has been shown that many genotypes circulate worldwide, all highly related to one of the two known lineages U/I and J. In this study, we evaluated the spatial and temporal distribution of CyHV-3 strains in a small enzootic area, the lake of Cirata (West Java, Indonesia). Of the 365 samples analysed, from clinical or asymptomatic fish, 244 were found positive for CyHV-3, suggesting a high occurrence of the virus. Genotyping of these viral specimens with a range of molecular markers revealed the presence of numerous haplotypes in the host population, all related to the J lineage. In single individuals, mixed-genotype infections occurred at high frequency. The present results demonstrate that polymorphic molecular markers are suitable to monitor the genetic evolution of a viral population in an enzootic area., (© 2012 Blackwell Publishing Ltd.)

A betanodavirus associated with a massive mortality was isolated from larvae of tilapia, Oreochromis niloticus, maintained in fresh water at 30 degrees C. Histopathology revealed vacuolation of the nervous system, suggesting an infection by a betanodavirus. The virus was identified by indirect fluorescent antibody test in the SSN1 cell line and further characterized by sequencing of a PCR product. Sequencing of the T4 region of the coat protein gene indicated a phylogenetic clustering of this isolate within the red-spotted grouper nervous necrosis virus type. However, the tilapia isolate formed a unique branch distinct from other betanodavirus isolates. The disease was experimentally reproduced by bath infection of young tilapia at 30 degrees C. The reservoir of virus at the origin of the outbreak remains unidentified. To our knowledge, this is the first report of natural nodavirus infection in tilapia reared in fresh water.

Massive mortalities of Carassius auratus (L.) occurred in a farm in France during summer 2014. Fish presented anorexia, loss of scales and large amounts of mucus on the gills. Necrosis of the distal tip of the filament and the lamellae, combined with fusion of the lamellae, was observed, as well as necrosis in the hematopoietic organs and in the digestive tract. The histological examination led to hypothesize the implication of a virus in the mortality. The presence of cyprinid herpesvirus 2 (Cy HV-2) in dead fish was demonstrated by amplification and sequencing of portions of the DNA polymerase and helicase genes, both sequences exhibiting 100% identity with Cy HV-2 from Japan. In an attempt to find genetic markers of variation, two regions containing tandem repeats in the Japanese genome were amplified from a virus-positive sample from the present outbreak. A first region ( mB) was fully identical to the Japanese isolate. However, the second region ( mA) exhibited a range of deletions and substitutions compared to Cy HV-2 from Japan. This is the first report of Cy HV-2 in France in association with mortality of goldfish and the first identification of a molecular marker for its tracing. [ABSTRACT FROM AUTHOR]