Emergence of carp edema virus (CEV) and its significance to European common carp and koi Cyprinus carpio.

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.

Molecular identification of iridoviruses infecting various sturgeon species in Europe.

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.

First detection of Cyprinid Herpesvirus 2 (CyHV-2) in goldfish (Carassius auratus) in France.

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.

Fishpathogens.eu/noda: a free and handy online platform for Betanodavirus targeted research and data sharing.

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.

First isolation of hirame rhabdovirus from freshwater fish in Europe.

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.

Spatio-temporal analysis of cyprinid herpesvirus 3 genetic diversity at a local scale.

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.

Outbreak of betanodavirus infection in tilapia, Oreochromis niloticus (L.), in fresh water.

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.

[Animal circoviruses and associated diseases]. / Circovirus et pathologies associées chez les animaux.

Animal circoviruses belong to the Circovirus genus of the Circoviridae family. Nowadays, only swine and birds were identified as circovirus hosts. Circoviruses have a single-stranded circular genome of approximately 2000 nucleotide long. DNA of these viruses possesses : (i) a nonanucleotide sequence essential for replication, flanked by inverted repeat sequences, a palindrome that has the potential to form a stem-loop structure and (ii) two major ORFs, located on the viral and complementary strands, which encode respectively the replication-associated protein (Rep) and the capsid protein (Cap). All the circoviruses described at the present time, except porcine circovirus of type 1, are associated with immunosuppressive or immunodepressive diseases. Histopathological lesions such as cytoplasmic inclusions of virus in histiocytic cells and T and B lymphocyte depletion in lymphoid organs, are commonly noticed. No medical prophylaxis of circovirus infections is currently available.

First generic one step real-time Taqman RT-PCR targeting the RNA1 of betanodaviruses.

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.

Differentiation between Cyprinid herpesvirus type-3 lineages using duplex PCR.

To date, all the isolates of Cyprinid herpesvirus type-3 (CyHV3) responsible for serious outbreaks in carps Cyprinus carpio have been found to be very similar or identical on the basis of DNA sequences of a few reference genes. However, two genetic lineages (U/I and J) are distinguished by full-length genome sequencing. Two molecular markers presenting genetic variations were targeted for developing a duplex PCR assay able to distinguish CyHV3-U/I from CyHV3-J while avoiding DNA sequencing. The method was validated on a series of 42 samples of infected carps from France, The Netherlands and Poland collected from 2001 to 2008. Among these samples, both the U/I and J genotypes were identified, but also a third genotype representing a genetic intermediate between U/I and J for one of the two molecular markers. A classification of CyHV3 genotypes, based on the alleles of the two molecular markers, is proposed. The assay is easy to perform and provides a genotype information with samples moderately or highly concentrated. This tool should improve our knowledge regarding the present distribution and future diversification of this emerging virus.

Pig anelloviruses are highly prevalent in swine herds in France.

A survey of anelloviruses in swine herds from Britanny, France, is reported. By using PCR targeted to the conserved untranslated region, prevalences of 93 and 73 % were found among 15 herds and 33 animals, respectively. The lung was the organ found to be positive most frequently among the five organs tested from 32 animals. The highest identity levels of our nucleotide sequences were found with pig isolates from Japan and with an isolate from Tupaia belangeri. Interestingly, when aligning all available swine isolates from France and Japan, at least two phylogenetic groups were identified, each one containing clones from France and Japan. Some animals carried clones from both groups, demonstrating intra-individual variability. Despite the putative harmlessness of anelloviruses, the potential inoculum carried by pigs must be further evaluated as a sanitary threat.

Nucleotide sequence evidence for three distinct sugarcane streak mastreviruses.

The complete sequences of four clones of sugarcane streak virus (SSV) isolates from Egypt and one SSV clone from Reunion island were determined. The four Egyptian genomes were highly similar to one another (97-99% nt identity) and were considered as variants of the same virus. The Egyptian SSV was genetically different from all other mastreviruses, the closest virus being SSV from South-Africa (60% nt identity), and defined as a new mastrevirus species named SSEV. The SSV clone from Reunion was highly related to the SSV from Mauritius and SSV from Nigeria, for which only partial sequences were available, indicating that the three sugarcane streak isolates from Mauritius, Reunion and Nigeria were strains of the same virus tentatively named SSMV. This work further confirms that SSMV is a distinct viral species compared to other mastreviruses, including the SSEV (59% nt identity) and SSV (66% nt identity). By comparing two clones from the Mascarene islands, we correlated substitutions in the C-terminal end of the coat protein with a different response to a monoclonal antibody, providing data on the mapping of a specific epitope. Agroinoculations experiments demonstrated that an SSEV clone induced more severe symptoms on maize than two clones from the Mascarene. Inside the African streak virus cluster, the sugarcane mastrevirus isolates were gathered in a sub-cluster of three viruses, SSEV, SSV and SSMV. The diversity of the SSVs is discussed in relation to its host, sugarcane, an imported crop in Africa.