Genomic and Phenotypic Characterization of a Novel Virulent Strain of 2 Originating from an Outbreak in The Netherlands.

2 (CyHV-2) is the causative agent of herpesviral hematopoietic necrosis in several economically important farmed freshwater fish species of the genus . Despite several CyHV-2 strains being isolated and fully sequenced, there is a lack of detailed characterization and consistent information on strains that exhibit high virulence in adult goldfish through viral challenge by immersion, particularly in the context of European strains and host populations. Strains that can cause highly virulent disease via this inoculation route are much more compatible with experimental designs that are representative of natural infection; thus, their utilization provides greater biological relevance. Consequently, in this study, we isolated three novel strains of CyHV-2 (designated NL-1, NL-2, and NL-3), originating from outbreaks in The Netherlands. Full-length genome sequencing and phylogenetic analyses revealed that these newly isolated strains are distinct from known strains and from each other. Significant differences were observed between the strains, in terms of in vitro growth kinetics, with NL-2 exhibiting stable passaging and superior fitness in vitro. Importantly, the challenge of adult Shubunkin goldfish with the NL-2 strain via immersion (2000 PFU/mL) induced an average mortality of ~40%, while parallel experiments with the CyHV-2 reference strain ST-J1 resulted in no mortality. Taken together, this study represents the characterization of a new CyHV-2 in vivo infection model, much more compatible with experimental designs that are required to be representative of natural infection. This model will be extremely useful in many aspects of CyHV-2 research in the future. Importantly, the genetic and phenotypic characterization performed in this study generates hypotheses on the potential roles of CyHV-2 genes in adaptation of the virus in vitro or in vivo.