title: Targeted genomic enrichment and sequencing of CyHV-3 from carp tissues confirms low nucleotide diversity and mixed genotype infections.
abstract: Koi herpesvirus disease (KHVD) is an emerging disease that causes mass mortality in koi and common carp,Cyprinus carpioL. Its causative agent is Cyprinid herpesvirus 3 (CyHV-3), also known as koi herpesvirus (KHV). Although data on the pathogenesis of this deadly virus is relatively abundant in the literature, still little is known about its genomic diversity and about the molecular mechanisms that lead to such a high virulence. In this context, we developed a new strategy for sequencing full-length CyHV-3 genomes directly from infected fish tissues. Total genomic DNA extracted from carp gill tissue was specifically enriched with CyHV-3 sequences through hybridization to a set of nearly 2 million overlapping probes designed to cover the entire genome length, using KHV-J sequence (GenBank accession number AP008984) as reference. Applied to 7 CyHV-3 specimens from Poland and Indonesia, this targeted genomic enrichment enabled recovery of the full genomes with >99.9% reference coverage. The enrichment rate was directly correlated to the estimated number of viral copies contained in the DNA extracts used for library preparation, which varied between ∼5000 and ∼2×107. The average sequencing depth was >200 for all samples, thus allowing the search for variants with high confidence. Sequence analyses highlighted a significant proportion of intra-specimen sequence heterogeneity, suggesting the presence of mixed infections in all investigated fish. They also showed that inter-specimen genetic diversity at the genome scale was very low (>99.95% of sequence identity). By enabling full genome comparisons directly from infected fish tissues, this new method will be valuable to trace outbreaks rapidly and at a reasonable cost, and in turn to understand the transmission routes of CyHV-3.
authors.affiliation: Institut des Sciences de l'Evolution de Montpellier, UMR226 IRD-CNRS-UM-EPHE , Montpellier , France.
authors.forename: Saliha
authors.initials: S
authors.lastname: Hammoumi
authors.affiliation: Université de Montpellier , Montpellier , France.
authors.forename: Tatiana
authors.initials: T
authors.lastname: Vallaeys
authors.affiliation: Main Center for Freshwater Aquaculture Development , Sukabumi , Indonesia.
authors.forename: Ayi
authors.initials: A
authors.lastname: Santika
authors.affiliation: Plate-forme Genotoul Bioinfo, UR875 Biométrie et Intelligence Artificielle, Institut National de la Recherche Agronomique , Castanet-Tolosan , France.
authors.forename: Philippe
authors.initials: P
authors.lastname: Leleux
authors.affiliation: Department of Fish Diseases, National Veterinary Research Institute , Pulawy , Poland.
authors.forename: Ewa
authors.initials: E
authors.lastname: Borzym
authors.affiliation: Plate-forme Genotoul Bioinfo, UR875 Biométrie et Intelligence Artificielle, Institut National de la Recherche Agronomique , Castanet-Tolosan , France.
authors.forename: Christophe
authors.initials: C
authors.lastname: Klopp
authors.affiliation: Institut des Sciences de l'Evolution de Montpellier, UMR226 IRD-CNRS-UM-EPHE , Montpellier , France.
authors.forename: Jean-Christophe
authors.initials: JC
authors.lastname: Avarre
keywords: Cyprinid herpesvirus 3; Genome; Mixed infection; Targeted genomic enrichment; Variability