Evaluation of tangential flow filtration coupled to long-read sequencing for ostreid herpesvirus type 1 genome assembly.

Whole-genome sequencing is widely used to better understand the transmission dynamics, the evolution and the emergence of new variants of viral pathogens. This can bring crucial information to stakeholders for disease management. Unfortunately, aquatic virus genomes are usually difficult to characterize because most of these viruses cannot be easily propagated in vitro. Developing methodologies for routine genome sequencing of aquatic viruses is timely given the ongoing threat of disease emergence. This is particularly true for pathogenic viruses infecting species of commercial interest that are widely exchanged between production basins or countries. For example, the ostreid herpesvirus type 1 (OsHV-1) is a Herpesvirus widely associated with mass mortality events of juvenile Pacific oyster Crassostrea gigas. Genomes of Herpesviruses are large and complex with long direct and inverted terminal repeats. In addition, OsHV-1 is unculturable. It therefore accumulates several features that make its genome sequencing and assembly challenging. To overcome these difficulties, we developed a tangential flow filtration (TFF) method to enrich OsHV-1 infective particles from infected host tissues. This virus purification allowed us to extract high molecular weight and high-quality viral DNA that was subjected to Illumina short-read and Nanopore long-read sequencing. Dedicated bioinformatic pipelines were developed to assemble complete OsHV-1 genomes with reads from both sequencing technologies. Nanopore sequencing allowed characterization of new structural variations and major viral isomers while having 99,98 % of nucleotide identity with the Illumina assembled genome. Our study shows that TFF-based purification method, coupled with Nanopore sequencing, is a promising approach to enable in field sequencing of unculturable aquatic DNA virus.

Genetic diversity and connectivity of the Ostreid herpesvirus 1 populations in France: A first attempt to phylogeographic inference for a marine mollusc disease.

The genetic diversity of viral populations is a key driver of the spatial and temporal diffusion of viruses; yet, studying the diversity of whole genomes from natural populations still remains a challenge. Phylodynamic approaches are commonly used for RNA viruses harboring small genomes but have only rarely been applied to DNA viruses with larger genomes. Here, we used the Pacific oyster mortality syndrome (a disease that affects oyster farms around the world) as a model to study the genetic diversity of its causative agent, the Ostreid herpesvirus 1 (OsHV-1) in the three main French oyster-farming areas. Using ultra-deep sequencing on individual moribund oysters and an innovative combination of bioinformatics tools, we de novo assembled twenty-one OsHV-1 new genomes. Combining quantification of major and minor genetic variations, phylogenetic analysis, and ancestral state reconstruction of discrete traits approaches, we assessed the connectivity of OsHV-1 viral populations between the three oyster-farming areas. Our results suggest that the Marennes-Oléron Bay represents the main source of OsHV-1 diversity, from where the virus has dispersed to other farming areas, a scenario consistent with current practices of oyster transfers in France. We demonstrate that phylodynamic approaches can be applied to aquatic DNA viruses to determine how epidemiological, immunological, and evolutionary processes act and potentially interact to shape their diversity patterns.

Monitoring Autophagy at Cellular and Molecular Level in Crassostrea gigas During an Experimental Ostreid Herpesvirus 1 (OsHV-1) Infection.

Mortality outbreaks of young Pacific oysters, Crassostrea gigas, have seriously affected the oyster-farming economy in several countries around the world. Although the causes of these mortality outbreaks appear complex, a viral agent has been identified as the main factor: a herpesvirus called ostreid herpesvirus 1 (OsHV-1). Autophagy is an important degradation pathway involved in the response to several pathologies including viral diseases. In C. gigas, recent studies indicate that this pathway is conserved and functional in at least haemocytes and the mantle. Furthermore, an experimental infection in combination with compounds known to inhibit or induce autophagy in mammals revealed that autophagy is involved in the response to OsHV-1 infection. In light of these results, the aim of this study was to determine the role of autophagy in the response of the Pacific oyster to infection by virus OsHV-1. For this purpose, an experimental infection in combination with a modulator of autophagy was performed on Pacific oysters known to have intermediate susceptibility to OsHV-1 infection. In haemolymph and the mantle, the autophagy response was monitored by flow cytometry, western blotting, and real-time PCR. At the same time, viral infection was evaluated by quantifying viral DNA and RNA amounts by real-time PCR. Although the results showed activation of autophagy in haemolymph and the mantle 14 hours post infection (after viral replication was initiated), they were also indicative of different regulatory mechanisms of autophagy in the two tissues, thus supporting an important function of autophagy in the response to virus OsHV-1.

Genomic Diversity of the Ostreid Herpesvirus Type 1 Across Time and Location and Among Host Species.

The mechanisms underlying virus emergence are rarely well understood, making the appearance of outbreaks largely unpredictable. This is particularly true for pathogens with low per-site mutation rates, such as DNA viruses, that do not exhibit a large amount of evolutionary change among genetic sequences sampled at different time points. However, whole-genome sequencing can reveal the accumulation of novel genetic variation between samples, promising to render most, if not all, microbial pathogens measurably evolving and suitable for analytical techniques derived from population genetic theory. Here, we aim to assess the measurability of evolution on epidemiological time scales of the Ostreid herpesvirus 1 (OsHV-1), a double stranded DNA virus of which a new variant, OsHV-1 µVar, emerged in France in 2008, spreading across Europe and causing dramatic economic and ecological damage. We performed phylogenetic analyses of heterochronous (n = 21) OsHV-1 genomes sampled worldwide. Results show sufficient temporal signal in the viral sequences to proceed with phylogenetic molecular clock analyses and they indicate that the genetic diversity seen in these OsHV-1 isolates has arisen within the past three decades. OsHV-1 samples from France and New Zealand did not cluster together suggesting a spatial structuration of the viral populations. The genome-wide study of simple and complex polymorphisms shows that specific genomic regions are deleted in several isolates or accumulate a high number of substitutions. These contrasting and non-random patterns of polymorphism suggest that some genomic regions are affected by strong selective pressures. Interestingly, we also found variant genotypes within all infected individuals. Altogether, these results provide baseline evidence that whole genome sequencing could be used to study population dynamic processes of OsHV-1, and more broadly herpesviruses.