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

Abstract

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 differentiation and host specialization among OsHV-1 infecting two oyster species in France

“…However, it is plausible that recombination or rearrangement events, which are common in herpesviruses, play a crucial role in their appearance. Similarly, studies based on both Sanger sequencing [12] and recently Nanopore long reads sequencing [51] have demonstrated the presence of four major genomic isomers of the OsHV-1 genome, considered as structural variations, that were present in stoichiometric proportions. In herpesviruses, and especially in the herpes simplex virus 1, the presence of isomeric genomes is closely related to the replication and recombination mechanisms [87][88][89][90] and occurs early during the herpesvirus life cycle.…”

“…To avoid inverted repeat regions reconstruction issues for all subsequent analyses, every OsHV-1 genome was reconstructed as non-redundant (NR) genome containing only one copy of each duplicated regions (IRL and IRS) as described by [60], [50] and [51]. NR-genomes were validated and corrected by similarity matrix visualization of each genome to itself using the dotplot tool (based on the EMBOSS v6.5.7 tool dottup) implemented in Geneious Prime v2022.2.2.…”

“…As the minimum threshold for efficient Illumina sequencing of virus particles is 10 3 copies, all samples were found to be suitable for sequencing [37,50,51].…”

Advances in Viral Aquatic Animal Disease Knowledge: The Molecular Methods’ Contribution

“…So, due to the ongoing threat of disease emergence, it is essential to develop methodologies for the routine genome sequencing of aquatic viruses. This is especially true for pathogenic viruses that infect species of commercial interest that are frequently traded between production basins or countries [ 135 ].…”

“…With the above-described molecular procedures, some viruses, such as OsHV-1, can be difficult to be detected and identified when present in low amounts [ 127 ]. To enhance the recovery of OsHV-1 infectious particles from infected host tissues, a tangential flow filtration (TFF) technique was devised [ 135 ]. This method permitted a high molecular weight and high-quality viral DNA to be obtained for Illumina short-read and Nanopore long-read sequencing.…”