Aurélie Dotto-Maurel

The European eel has a complex life cycle involving various developmental stages and migrations through marine and freshwater environments. It is critically endangered and therefore a flagship species for conservation efforts. The European eel is also an important fish species in aquaculture. One of the major pathogens contributing to the decline of the European eel is thought to be anguillid herpesvirus 1 (AngHV-1). In this study, we used in vivo bioluminescent imaging to study the ability of AngHV-1 to infect the various developmental stages of the European eel, and to investigate virus transmission between eels. Our results highlight that eels become sensitive to AngHV-1 infection only when they become yellow eels (a freshwater stage prior to maturity), with the gills and the periodontal mucosa acting as the first sites of replication after inoculation by immersion in virus-containing water. Also, multifocal cutaneous bioluminescent signals appeared in grouped eels, but not in isolated eels. These observations suggested that the virus might be transmitted through biting between fish. In support of this hypothesis, experiments based on single artificial bites occurring between infected eels and naïve eels led to the infection of all exposed subjects. The biological relevance of AngHV-1 transmission by biting was further supported by observation of the instability of virion infectivity in water and fish mucus. Our study thus challenges the current paradigm that fish viruses are transmitted mainly via water acting as an abiotic vector. It demonstrates that, as in air, so in water, social interactions matter. IMPORTANCE: Fish viruses have devastating effects on wildlife and severe economic impact on aquaculture. The current paradigm is that they are transmitted via water acting as an abiotic vector. Here, we used in vivo bioluminescent imaging to study the ability of anguillid herpesvirus 1 (AngHV-1) to infect different developmental stages of European eel and to investigate virus transmission between eels. The major findings were that (i) eels become sensitive to AngHV-1 infection only when they become yellow eels, with the gills and the periodontal mucosa acting as the first sites of replication after inoculation by immersion in virus-containing water; (ii) AngHV-1 is efficiently transmitted between fish through biting, thereby avoiding virion instability in water and mucus. Our study thus challenges the current paradigm that fish viruses are transmitted mainly via water acting as an abiotic and protective vector. It demonstrates that, as in air, so in water, social interactions matter.

Depuis les années 1990, l’huître creuse Magallana gigas (anciennement Crassostrea gigas) connaît d’importantes mortalités aux stades larvaire et juvénile, associées à l’Ostreid herpesvirus de type 1 (OsHV-1). Ce virus à ADN double brin, appartenant à l’ordre des Herpesvirales et à la famille des Malacoherpesviridae. Le cycle de vie du virus OsHV-1 comporte deux phases : une phase lytique, responsable de mortalités massives du naissain et d’une forte excrétion de particules virales dans l’eau, et une potentielle phase de persistance, durant laquelle il se maintiendrait sous une forme encore indéterminée dans un réservoir non identifié. La structure des particules virales du virus OsHV-1 est similaire à celle des Orthoherpesviridae, et son organisation génomique, comparable à celle des Alphaherpesvirinae, comporte deux régions uniques encadrées de séquences répétées et inversées pouvant permuter et former des isomères génomiques. En raison de cette complexité génomique, les approches de séquençage à lectures courtes ne permettent pas un assemblage complet du génome viral ni l’identification précise des isomères. Dans ce contexte, cette thèse visait à : (i) évaluer les performances du séquençage à longues lectures Oxford Nanopore Technologies (ONT), (ii) caractériser la plasticité de l’architecture génomique et la dynamique transcriptionnelle du virus OsHV-1 en phase lytique M. gigas, et (iii) d’identifier les réservoirs potentiels du virus OsHV-1 durant sa phase de persistance et d’en caractériser les mécanismes moléculaires. Les résultats obtenus démontrent que la technologie ONT constitue un outil performant pour surmonter les limites du séquençage à lectures courtes. Ils révèlent également une dynamique d’expression génique en phase lytique similaire à celle d’autres herpèsvirus et apportent des éléments en faveur d’une persistance du virus sous forme latente dans le naissain de M. gigas. Ces travaux représentent une avancée importante dans la compréhension des interactions entre OsHV-1 et M. gigas, et ouvrent la voie à de nouvelles approches de gestion des maladies virales en ostréiculture.

Since the 1990s, the Pacific oyster ( Magallana gigas ) has experienced repeated mortality events associated with Ostreid herpesvirus 1 (OsHV-1). Although the virus has been genomically characterised, its replication cycle and its interactions with the oyster immune system are still not well understood. In particular, little is known about the dynamics of OsHV-1 gene expression and the immune responses of haemocytes from oysters with varying susceptibility to the virus. While some studies have focused on the expression of specific viral and host genes in whole oysters, none have provided a comprehensive analysis of genome-wide expression across multiple post-infection time points in haemocytes. The lack of oyster cell lines makes studying virus–host interactions in vitro challenging. However, haemocytes, the key immune cells circulating in haemolymph, can be maintained in vitro in the short term and represent a relevant model for analysing infection dynamics. In this study, haemocytes from two M. gigas batches, one highly susceptible and one less susceptible to OsHV-1, were infected in vitro . We tracked the viral and host transcriptomes over a 24-h period post-infection using high-throughput dual transcriptomics. Our results provide a detailed overview of the OsHV-1 transcriptomic landscape in haemocytes from high- and low-susceptible M. gigas over time. In addition, weighted correlation network analysis of host gene expression provided insights into the haemocytes’ response to infection and highlighted batch-specific immune responses. This comprehensive transcriptomic study is the first to describe virus–host interactions across multiple stages of infection in haemocytes from Pacific oysters, showing contrasted survival when exposed to OsHV-1.

Since the 1990s, the Pacific oyster Magallana gigas has faced significant mortality, which has been associated with the detection of the Ostreid Herpesvirus type 1 (OsHV-1). Due to the complex genomic architecture and the presence of multiple genomic isomers, short-read sequencing using Illumina method struggles to accurately assemble tandem and repeat regions and to identify and characterize large structural variations in the OsHV-1 genome. Third-generation sequencing technologies, as long-read real-time nanopore sequencing from Oxford Nanopore Technologies (ONT), offer new possibilities for OsHV-1 whole-genome analysis. Identification of the best method for extraction of high molecular weight (HMW) DNA and development of accurate bioinformatic pipelines for its characterization are now required. To this end, we evaluated and compared six HMW methods and one conventional DNA extraction kit for their ability to extract OsHV-1 DNA from M. gigas -infected tissues. We then evaluated the ability of ONT sequencing to produce an accurate OsHV-1 genome from both whole-genome and “adaptive sampling” (AS) sequencing approaches. Finally, we evaluated the efficiency of bioinformatics tools for de novo assembly and consensus calling to generate accurate OsHV-1 genomes. The HMW DNA extraction kit coupled with ONT sequencing and dedicated bioinformatics tools allowed us to produce accurate OsHV-1 genomes compared to those assembled using Illumina technology. The AS approach allowed up to 60% enrichment for viral data, and the long reads generated by ONT allowed the characterization of OsHV-1 isomers. Together with its portability, this sequencing shows great promise as a diagnostic tool for the characterization of unculturable aquatic viruses directly from host tissues. IMPORTANCE: Many aquatic viruses threaten commercially valuable species and cause significant economic losses during outbreaks. To improve our understanding of the origin, transmission patterns and spread of these viruses, additional genomic data are essential. However, genomic characterization of unculturable large DNA viruses is a major challenge. In the present study, we have successfully evaluated the ability of ONT sequencing and adaptive sequencing (AS) to sequence and assemble the complete OsHV-1 genome. Our results show that it is now possible to sequence the whole genome of large DNA viruses directly from infected host tissue, without the need for prior in vitro propagation or prior laboratory steps for virus enrichment.