Joëlle Cabon

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.

Effect of chronic exposure to pendimethalin on the susceptibility of rainbow trout, Oncorhynchus mykiss L., to viral hemorrhagic septicemia virus (VHSV).

In this study, the in vivo effects of chronic pollution by the active substance (AS) pendimethalin, a dinitroaniline herbicide, on the susceptibility of rainbow trout, Oncorhynchus mykiss L., to an experimental challenge with viral hemorrhagic septicemia virus (VHSV) were assessed. After four weeks of exposure to fresh water (C group) or 500 ng L(-1) of AS (P500 group), the fish were challenged by immersion in water containing 10(4) TCID(50) mL(-1) of VHSV. While exposure to pendimethalin was maintained throughout the experiment, mortalities were recorded during the 40 days post-infection (dpi) and organs were collected from dead fish for virological examination. At the end of the experiment, anti-VHSV antibodies and the classical pathway of complement activity were assessed in trout plasma. Exposure to pendimethalin significantly affected the distribution of cumulative mortality accelerating death in fish infected by VHSV. Pendimethalin appeared to decrease the Mean Time to Death (MTD) after virus treatment from 14.9 days (C-VHSV) to 10.2 days (P500-VHSV). Nevertheless, by the end of the experiment, differences in cumulative mortality were no longer observed between the two groups, which had reached the same stage (50 percent). Furthermore, a higher concentration of the virus was recovered from the pools of organs from the P500-VHSV group than the C-VHSV group. Moreover, at 40 dpi, although no significant difference was observed in the immune response between the two groups, more fish in the P500-VHSV group had set up an immune response by secreting antibodies than in the control viral group (C-VSHV).

First isolation of a rhabdovirus from perch Perca fluviatilis in Switzerland.

Perca fluviatilis is a fish species of increasing interest to the Swiss fish farming industry. In recent years, recirculation systems have been specifically set up to increase production. In one of these farms, abnormal spiral swimming associated with elevated mortalities occurred in repeated batches of imported perch shortly after stocking on several occasions. No bacterial or parasitic etiology was detected, but a virus grown in bluegill fry (BF-2) cells was identified as perch rhabdovirus. Subsequent investigations of other samples suggested a viral tropism for the central nervous system (CNS). Phylogenetic analysis of the partial N and entire G gene sequences positioned this isolate in genogroup C of the species Perch rhabdovirus, with high nucleotide and amino acid (aa) sequence identities with the DK5533 strain isolated in Denmark in 1989. Comparative studies using other closely related isolates allowed the distinction of 2 serological patterns among perch rhabdoviruses and the identification of a proline substitution by a serine in position 147 of the glycoprotein potentially involved in antigenic differentiation. Even if perch imported onto the farm tested negative by virus isolation prior to transport, they may have been the origin of this outbreak since CNS tissue was not included in the samples that were analyzed. Another possibility might be a sub-clinical infection with a viral load in resident fish too low to be detected. This study reports the first isolation of a perch rhabdovirus in Switzerland, and emphasizes the necessity of optimizing diagnostic tools that facilitate better control of the risks associated with fish translocation.

A single amino acid change in the non-structural NV protein impacts the virulence phenotype of Viral hemorrhagic septicemia virus in trout

Novirhabdoviruses like the Viral hemorrhagic septicemia virus (VHSV) are rhabdoviruses infecting fish. In the current study, RNA genomes of different VHSV field isolates classified as high, medium or low virulent phenotypes have been sequenced by next-generation sequencing and compared. Various amino acid changes, depending on the VHSV phenotype, have been identified in all the VHSV proteins. As a starting point, we focused our study on the non-virion (NV) non-structural protein in which an arginine residue (R116) is present in all the virulent isolates and replaced by a serine/asparagine residue S/N116 in the attenuated isolates. A recombinant virus derived from a virulent VHSV strain in which the NV R116 residue has been replaced by a serine, rVHSVNVR116S, was generated by reverse genetics and used to infect juvenile trout. We showed that rVHSVNVR116S was highly attenuated and that surviving fish were almost completely protected from a challenge with the wild-type VHSV.

Comparative biomarker responses in Japanese medaka (Oryzias latipes) exposed to benzo[a]pyrene and challenged with betanodavirus at three different life stages

It is now well documented that several contaminants can modulate the fish immune system, leading to disrupted host resistance against pathogens and increased incidence of disease. Since fish are usually co-exposed to chemicals and pathogens in the natural environment, analysis of the immunotoxic effects of pollutants is particularly relevant. The authorities in the European Union have recommended the development of toxicity assays on cell cultures and embryos, as an alternative to testing in vertebrates. This is why in our study, a fish immune challenge assay was developed for the early life stages of Japanese medaka to evaluate and compare the relevance of new biomarkers. Fish were exposed to benzo[a]pyrene (BaP), a model pollutant, for 8days at the embryonic stage, or for 48h at the larvae and juvenile stages, and fish were infected with betanodavirus by bath-challenge of 106TCID50/mL. Biometric changes and induction of malformations were observed after embryonic exposure. DNA damage and induction of EROD activity were recorded at the end of all chemical exposures. Viral infection increased the mortality rate significantly and disturbed the behavior of fish after light stimulation. While BaP exposure increased swimming speed, betanodavirus infection slowed swimming activity. In larvae co-exposed to BaP and the virus, the viral titer in the whole body was higher than in fish infected only with the virus. This study highlighted the sensitivity and usefulness of the immune challenge assay on the early life stages of Japanese medaka to evaluate the toxic effects of pollutants.

Transchem project – Part II: Transgenerational effects of long-term exposure to pendimethalin at environmental concentrations on the early development and viral pathogen susceptibility of rainbow trout ( Oncorhynchus mykiss)

In the Transchem project, rainbow trout genitors were exposed to environmental concentrations of pendimethalin over a period of 18 months and two new first generations of offspring, F1_2013 and F1_2014, were obtained. We investigated the impact of direct chemical exposure on juveniles as well as the potential cumulative transgenerational and direct effects on the larval development and on the pathogen susceptibility of offspring. Depending on the chemical treatment or not of the adults, their offspring were distributed in the tanks of our experimental system, in two batches i.e. juveniles from the control genitors (G-) and others from the contaminated ones (G+), and then, half of the tanks were exposed daily to pendimethalin (Off+) while the others were used as controls (Off-). Viral challenges were performed on the offspring, before and after three months of direct chemical exposure, with strains of infectious hematopoietic necrosis virus (IHNV), viral haemorrhagic septicemia virus (VHSV) and sleeping disease alphavirus (SDV). Direct and transgenerational macroscopic effects were observed on offspring, with a percentage of abnormalities in offspring derived from the genitors exposed to pendimethalin (G+) significantly higher compared to those from the genitors from non-exposed group (G-). Before the direct chemical exposure, similar kinetics of mortality was observed between the offspring from the contaminated or control genitors after VHSV infection. With IHNV, the G+ group died in a slightly larger proportion compared to the G- group and seroconversion was greater for the G- group. For the SDV challenge, the mortality was delayed for the G+ offspring compared to the G- and seroconversion reached 65% in the G+ group compared to 45% in the G-, with similar antibody titres. After three months of direct chemical exposure, kinetics of mortality induced by IHNV infection were similar for all groups studied. Infection with SDV resulted in a cumulative mortality of 40% for the G- groups (Off- and Off+), significantly higher than those observed from the contaminated genitors G+. Proportion of seropositivity for SDV varied from 24 to 47% depending on the group, with very low quantities of secreted antibodies. Lastly, the direct exposure of offspring could impact the capacity of fish to adapt their haematological parameters to environmental and physiological changes, and underlines the potential toxic effects on the next generations.