Characterization of a New Toti-like Virus in Sea Bass, Dicentrarchus labrax.

The European sea bass Dicentrarchus labrax is the main species reared in Mediterranean aquaculture. Its larval stage, which is very sensitive and highly affected by sanitary and environmental conditions, is particularly scrutinized in hatcheries. Recently, a Mediterranean sea bass farm had to deal with an abnormal increase in mortality, especially between 20 and 35 days post-hatching (dph). Biological investigations led to the observation of cytopathic effects on three different fish cell lines after almost 3 weeks of culture at 14 °C in contact with homogenized affected larvae, suggesting the presence of a viral agent. High-throughput sequencing revealed a 6818-nucleotide-long RNA genome with six putative ORFs, corresponding to the organization of viruses belonging to the Totiviridae family. This genome clustered with the newly described and suggested Pistolvirus genus, sharing 45.5% to 37.2% nucleotide identity with other piscine toti-like viruses such as Cyclopterus lumpus toti-like virus (CLuTLV) or piscine myocarditis virus (PMCV), respectively. Therefore, we propose to name this new viral agent sea bass toti-like virus (SBTLV). Specific real-time RT-PCR confirmed the presence of the viral genome in the affected larval homogenate from different production batches and the corresponding cell culture supernatant. Experimental infections performed on sea bass fingerlings did not induce mortality, although the virus could be detected in various organs and a specific immune response was developed. Additional studies are needed to understand the exact involvement of this virus in the mortality observed in hatcheries and the potential associated cofactors.

Characterization of a novel picornavirus isolated from moribund gilthead seabream (Sparus aurata) larvae.

Gilthead seabream represents a species of importance in Mediterranean aquaculture. The larval stage is particularly sensitive and frequently impacted in suboptimal environmental or sanitary conditions. In the present study, investigations were carried out in a seabream hatchery following an unusual mortality reaching 70% among 50-day post-hatching. Anorexia, loss of appetite and abnormal swimming behaviour were observed in absence of parasites or pathogenic bacteria. Proliferation of rod-shaped bacteria in the gut lumen was associated with focal degeneration in the intestinal mucosa. Cytopathic effects on an EK-1 cell line after 21 days of culture at 14°C and 20°C in contact with homogenized affected larvae revealed the presence of a viral agent. Molecular characterization by high-throughput sequencing showed a typical picornavirus genome organization with a polyprotein precursor of 2276 amino acids sharing 46.3% identity with that of the Eel Picornavirus-1. A specific real-time PCR confirmed the presence of the viral genome in affected larval homogenate and corresponding cell culture supernatant. We propose the name Potamipivirus daurada for this novel species within the genus Potamipivirus. The etiological role of this virus remains uncertain at this time, and future studies will be necessary to investigate its prevalence in natural and aquaculture-reared populations as well as its ability to cause diseases in gilthead seabream.

Changes in defense capacity to infectious hematopoietic necrosis virus (IHNv) in rainbow trout intergenerationally exposed to glyphosate.

Glyphosate, the most widely used herbicide active substance worldwide, has raised many scientific, political and public debates in the context of its recent re-registration in the European Union, highlighting in particular a lack of data concerning its potential generational effects. In this study, we investigated the intergenerational toxicity of this active substance used alone or coformulated in glyphosate-based herbicides (GBHs) on the ability of rainbow trout (Oncorhynchus mykiss) to face a viral challenge. Juvenile trout from parents exposed for eight months to four different chemical exposure conditions (non-exposed control, pure glyphosate, Roundup Innovert®, and Viaglif Jardin® were experimentally infected with the infectious hematopoietic necrosis virus (IHNv). Various enzymatic and hemato-immunological markers were assessed before and after the viral challenge. Chemical contamination with GBHs strongly modulated viral trout susceptibility. Pure glyphosate induced a cumulative mortality of 35.8%, comparable to the control (37.0%), which was significantly reduced with Roundup Innovert® (-9.9%) and increased (+14.8%) with Viaglif Jardin®. No modification was observed for the biomarkers analysed for any conditions. These results demonstrate that the nature of the co-formulants associated to glyphosate in GHBs can modulate the susceptibility of fish to pathogens.

Generational effects of a chronic exposure to a low environmentally relevant concentration of glyphosate on rainbow trout, Oncorhynchus mykiss.

In the past few decades, glyphosate became the most used herbicide substance worldwide. As a result, the substance is ubiquitous in surface waters. Concerns have been raised about its ecotoxicological impact, but little is known about its generational toxicity. In this study, we investigate the impact of an environmentally relevant concentration of glyphosate and its co-formulants on an F2 generation issued from exposed generations F0 and F1. Trans, inter and multigenerational toxicity of 1 µgL-1 of the active substance was evaluated on early stages of development and juvenile rainbow trout (Oncorhynchus mykiss) using different molecular, biochemical, immuno-hematologic, and biometric parameters, behavior analysis, and a viral challenge. Reproductive parameters of generation F1 were not affected. However, developmental toxicity in generation F2 due to glyphosate alone or co-formulated was observed with head size changes (e.g. head surface up to +10%), and metabolic disruptions (e.g. 35% reduction in cytochrome-c-oxidase). Moreover, larvae exposed transgenerationally to Viaglif and intergenerationally to glyphosate and Roundup presented a reduced response to light, potentially indicating altered escape behavior. Overall methylation was, however, not altered and further experiments using gene-specific DNA metylation analyses are required. After several months, biochemical parameters measured in juvenile fish were no longer impacted, only intergenerational exposure to glyphosate drastically increased the susceptibility of rainbow trout to hematopoietic necrosis virus. This result might be due to a lower antibody response in exposed fish. In conclusion, our results show that generational exposure to glyphosate induces developmental toxicity and increases viral susceptibility. Co-formulants present in glyphosate-based herbicides can modulate the toxicity of the active substance. Further investigations are required to study the specific mechanisms of transmission but our results suggest that both non-genetic mechanisms and exposure during germinal stage could be involved.

Comparative analysis of the course of infection and the immune response in rainbow trout (Oncorhynchus mykiss) infected with the 5 genotypes of infectious hematopoietic necrosis virus.

Infectious hematopoietic necrosis virus (IHNV) is a pathogen of importance for salmonid aquaculture. In this study, we aimed to characterize virus behavior and defense mechanisms developed in rainbow trout (RT, Oncorhynchus mykiss) experimentally infected with isolates belonging to the five described genotypes of IHNV, i.e. L, U, M, E and J. Mortality was monitored for two months, and blood and target organs were sampled at different times post-infection to assess viral load and cellular and humoral immune responses. Profiles of virulence were highly linked to precocious viral replication but also to the innate and specific immunity elicited in the host. Seroneutralization test (SNT) used for specific antibodies detection exhibited reliable results, with efficient cross-neutralization observed in heterologous systems except for the Asian representative. These data bring new insights about IHNV/RT interaction and reinforce the interest of SNT as preventive and epidemiological tool.

The Viral Hemorrhagic Septicemia Virus (VHSV) Markers of Virulence in Rainbow Trout (Oncorhynchus mykiss).

Viral hemorrhagic septicemia virus (VHSV) is a highly contagious virus leading to high mortality in a large panel of freshwater and marine fish species. VHSV isolates originating from marine fish show low pathogenicity in rainbow trout. The analysis of several nearly complete genome sequences from marine and freshwater isolates displaying varying levels of virulence in rainbow trout suggested that only a limited number of amino acid residues might be involved in regulating the level of virulence. Based on a recent analysis of 55 VHSV strains, which were entirely sequenced and phenotyped in vivo in rainbow trout, several amino acid changes putatively involved in virulence were identified. In the present study, these amino acid changes were introduced, alone or in combination, in a highly-virulent VHSV 23-75 genome backbone by reverse genetics. A total of 35 recombinant VHSV variants were recovered and characterized for virulence in trout by bath immersion. Results confirmed the important role of the NV protein (R116S) and highlighted a major contribution of the nucleoprotein N (K46G and A241E) in regulating virulence. Single amino acid changes in these two proteins drastically affect virus pathogenicity in rainbow trout. This is particularly intriguing for the N variant (K46G) which is unable to establish an active infection in the fins of infected trout, the main portal of entry of VHSV in this species, allowing further spread in its host. In addition, salmonid cell lines were selected to assess the kinetics of replication and cytopathic effect of recombinant VHSV and discriminate virulent and avirulent variants. In conclusion, three major virulence markers were identified in the NV and N proteins. These markers explain almost all phenotypes (92.7%) observed in trout for the 55 VHSV strains analyzed in the present study and herein used for the backward validation of virulence markers. The identification of VHSV specific virulence markers in this species is of importance both to predict the in vivo phenotype of viral isolates with targeted diagnostic tests and to improve prophylactic methods such as the development of safer live-attenuated vaccines.

VHSV Single Amino Acid Polymorphisms (SAPs) Associated With Virulence in Rainbow Trout.

The Viral Hemorrhagic Septicemia Virus (VHSV) is an OIE notifiable pathogen widespread in the Northern Hemisphere that encompasses four genotypes and nine subtypes. In Europe, subtype Ia impairs predominantly the rainbow trout industry causing severe rates of mortality, while other VHSV genotypes and subtypes affect a number of marine and freshwater species, both farmed and wild. VHSV has repeatedly proved to be able to jump to rainbow trout from the marine reservoir, causing mortality episodes. The molecular mechanisms regulating VHSV virulence and host tropism are not fully understood, mainly due to the scarce availability of complete genome sequences and information on the virulence phenotype. With the scope of identifying in silico molecular markers for VHSV virulence, we generated an extensive dataset of 55 viral genomes and related mortality data obtained from rainbow trout experimental challenges. Using statistical association analyses that combined genetic and mortality data, we found 38 single amino acid polymorphisms scattered throughout the complete coding regions of the viral genome that were putatively involved in virulence of VHSV in trout. Specific amino acid signatures were recognized as being associated with either low or high virulence phenotypes. The phylogenetic analysis of VHSV coding regions supported the evolution toward greater virulence in rainbow trout within subtype Ia, and identified several other subtypes which may be prone to be virulent for this species. This study sheds light on the molecular basis for VHSV virulence, and provides an extensive list of putative virulence markers for their subsequent validation.

Cellular, humoral and molecular responses in rainbow trout (Oncorhynchus mykiss) exposed to a herbicide and subsequently infected with infectious hematopoietic necrosis virus.

Aquatic ecosystems are now chronically polluted by a cocktail of many chemical substances. There is now clear evidence of associations between exposure to pollutants and greater susceptibility to pathogens. The aim of the present study was to characterize the defense capacities of rainbow trout (Oncorhynchus mykiss), chronically exposed to pendimethalin (PD), to subsequent experimental challenge with the infectious hematopoietic necrosis virus (IHNV). Immunological responses were examined at different organizational levels, from individuals to gene expression. No negative effects of PD were noted on the Fulton index nor on the liver or spleen somatic indices (LSI; SSI) before viral infection, but the infectious stress seems to generate a weak but significant decrease in Fulton and LSI values, which could be associated with consumption of energy reserves. During the viral challenges, the distribution of cumulative mortality was slightly different between infected groups. The impact of the virus on fish previously contaminated by PD started earlier and lasted longer than controls. The proportion of seropositive fish was lower in the fish group exposed to PD than in the control group, with similar quantities of anti-IHNV antibodies secreted in positive fish, regardless of the treatment. While no significant differences in C3-1 expression levels were detected throughout the experiment, TNF1&2, TLR3, Il-1ß and IFN expression levels were increased in all infected fish, but the difference was more significant in fish groups previously exposed to herbicide. On the other hand, ß-def expression was decreased in the pendimethalin-IHNV group compared to that in fish only infected by the virus (control-IHNV group).

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.

Molecular evolution and phylogeography of infectious hematopoietic necrosis virus with a focus on its presence in France over the last 30 years.

Infectious hematopoietic necrosis virus (IHNV) is among the most important pathogens affecting the salmonid industry. Here, we investigated the molecular evolution and circulation of isolates from 11 countries or regions all over the world, with a special focus on the epidemiological situation in France. The phylogeography, time to the most recent common ancestor (TMRCA) and nucleotide substitution rate were studied using 118 full-length glycoprotein gene sequences isolated from 9 countries (5 genogroups) over a period of 47 years. The TMRCA dates back to 1943, with the L genogroup identified as the likely root (67 %), which is consistent with the first report of this pathogen in the USA. A Bayesian inference approach was applied to the partial glycoprotein gene sequences of 88 representative strains isolated in France over the period 1987-2015. The genetic diversity of these 88 sequences showed mean nucleotide and amino-acid identities of 97.1 and 97.8 %, respectively, and a d N/d S ratio (non-synonymous to synonymous mutations) of 0.25, indicating purifying selection. The French viral populations are divided into eight sub-clades and four individual isolates, with a clear spatial differentiation, suggesting the predominant role of local reservoirs in contamination. The atypical 'signatures' of some isolates underlined the usefulness of molecular phylogeny for epidemiological investigations that track the spread of IHNV.

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.