Marine Baud

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.

Investigation of Cyprinid herpesvirus-3 genetic diversity by a multi-locus variable number of tandem repeats analysis

Cyprinid herpesvirus-3 (CyHV-3), or koi herpesvirus (KHV), is responsible for high mortalities in aquaculture of both common carp (Cyprinus carpio carpio) and koi carp (Cyprinus carpio koi) worldwide. The complete genomes of three CyHV-3 isolates showed more than 99% of DNA sequence identity, with the majority of differences located in short tandem repeats, also called VNTR (variable number of tandem repeats). By targeting these variations, eight loci were selected for genotyping CyHV-3 by multiple locus VNTR analysis (MLVA). CyHV-3 strains obtained after sequential in vivo infections exhibited identical MLVA profiles, whereas samples originating from a single isolate passaged 6 and 82 times in vitro exhibited mutations in two of the eight loci, suggesting a relatively slow genetic evolution rate of the VNTRs. The method was subsequently applied on 38 samples collected in Indonesia, France and the Netherlands. Globally, the isolates grouped in two main genetic clusters, each one divided in two subgroups including either CyHV-3-U/I or CyHV3-J. Interestingly, Indonesian strains were rather distant from CyHV-3-J isolate. The results of the present study indicate that these VNTR molecular markers are efficient in estimating the genetic diversity among CyHV-3 isolates and are therefore suitable for further molecular epidemiological studies.

Genetic diversity of perch rhabdoviruses isolates based on the nucleoprotein and glycoprotein genes

Despite the increasing impact of rhabdoviruses in European percid farming, the diversity of the viral populations is still poorly investigated. To address this issue, we sequenced the partial nucleoprotein (N) and complete glycoprotein (G) genes of nine rhabdoviruses isolated from perch (Perca fluviatilis) between 1999 and 2010, mostly from France, and analyzed six of them by immunofluorescence antibody test (IFAT). Using two rabbit antisera raised against either the reference perch rhabdovirus (PRhV) isolated in 1980 or the perch isolate R6146, two serogroups were distinguished. Meanwhile, based on partial N and complete G gene analysis, perch rhabdoviruses were divided into four genogroups, A-B-D and E, with a maximum of 32.9% divergence (G gene) between isolates. A comparison of the G amino acid sequences of isolates from the two identified serogroups revealed several variable regions that might account for antigenic differences. Comparative analysis of perch isolates with other rhabdoviruses isolated from black bass, pike-perch and pike showed some strong phylogenetic relationships, suggesting cross-host transmission. Similarly, striking genetic similarities were shown between perch rhabdoviruses and isolates from other European countries and various ecological niches, most likely reflecting the circulation of viruses through fish trade as well as putative transfers from marine to freshwater fish. Phylogenetic relationships of the newly characterized viruses were also determined within the family Rhabdoviridae. The analysis revealed a genetic cluster containing only fish viruses, including all rhabdoviruses from perch, as well as siniperca chuatsi rhabdovirus (SCRV) and eel virus X (EVEX). This cluster was distinct from the one represented by spring viraemia of carp vesiculovirus (SVCV), pike fry rhabdovirus (PFRV) and mammalian vesiculoviruses. The new genetic data provided in the present study shed light on the diversity of rhabdoviruses infecting perch in France and support the hypothesis of circulation of these viruses between other hosts and regions within Europe.

Molecular epidemiology of betanodaviruses isolated from sea bass and sea bream cultured along the Tunisian coasts

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.

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.

Phylogeny of the Viral Hemorrhagic Septicemia Virus in European Aquaculture.

One of the most valuable aquaculture fish in Europe is the rainbow trout, Oncorhynchus mykiss, but the profitability of trout production is threatened by a highly lethal infectious disease, viral hemorrhagic septicemia (VHS), caused by the VHS virus (VHSV). For the past few decades, the subgenogroup Ia of VHSV has been the main cause of VHS outbreaks in European freshwater-farmed rainbow trout. Little is currently known, however, about the phylogenetic radiation of this Ia lineage into subordinate Ia clades and their subsequent geographical spread routes. We investigated this topic using the largest Ia-isolate dataset ever compiled, comprising 651 complete G gene sequences: 209 GenBank Ia isolates and 442 Ia isolates from this study. The sequences come from 11 European countries and cover the period 1971–2015. Based on this dataset, we documented the extensive spread of the Ia population and the strong mixing of Ia isolates, assumed to be the result of the Europe-wide trout trade. For example, the Ia lineage underwent a radiation into nine Ia clades, most of which are difficult to allocate to a specific geographic distribution. Furthermore, we found indications for two rapid, large-scale population growth events, and identified three polytomies among the Ia clades, both of which possibly indicate a rapid radiation. However, only about 4% of Ia haplotypes (out of 398) occur in more than one European country. This apparently conflicting finding regarding the Europe-wide spread and mixing of Ia isolates can be explained by the high mutation rate of VHSV. Accordingly, the mean period of occurrence of a single Ia haplotype was less than a full year, and we found a substitution rate of up to 7.813 × 10−4 nucleotides per site per year. Finally, we documented significant differences between Germany and Denmark regarding their VHS epidemiology, apparently due to those countries’ individual handling of VHS.

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.