Illumination of Cyprinid herpesvirus 2 pathogenesis in both its natural host and a laboratory model

Cyprinid Herpesvirus 21 (CyHV-2), is a linear double-stranded DNA virus that belongs to the genus Cyprinivirus1 within the family Alloherpesviridae. This pathogenic agent is responsible for caus-ing high mortality infectious disease in goldfish (Carassius auratus), and other economically important Carassius spp. Despite its prevalence, a comprehensive investigation into host susceptibility and per-missivity at various developmental stages remains elusive, and the primary portal of viral entry into the host organism remains unclear. Also, unlike related viruses, the potential exploitation of zebrafish in-fection models, has not been explored. Furthermore, 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 host populations. This last point limits the study of CyHV-2 pathogenesis in the useful biological contexts most relevant to disease mitigation and control.
In order to address these knowledge gaps, we conducted a series of three interconnected studies, corresponding to Experimental Sections 1, 2 and 3 in this thesis. In the first study, we demonstrated that goldfish exhibit different levels of susceptibility and permissivity to the CyHV-2 virus at various devel-opmental stages and identified the primary portal for CyHV-2 entry into the host. This was achieved using a recombinant CyHV-2 strain expressing reporter genes. However, due to the parental strain for which the recombinant was derived from, it did not cause clinical disease in adult hosts when challenged by immersion, and thus it was not representative of highly pathogenic CyHV-2 strains responsible for high mortality outbreaks in the same host populations in the field. Therefore, in order to potentially establish alternative CyHV-2 in vivo infection models, we explored two avenues in parallel. Firstly (Experimental Section 2) we explored the possibility of establishing, through genetic engineering, new zebrafish strains, with increased susceptibility and permissively to CyHV-2. Secondly (Experimental Section 3) we sought to identify novel CyHV-2 strains that were highly virulent and capable of inducing CyHV-2 related mortality in European sourced adult goldfish when challenged via immersion. These three studies are summarized as follows in the format of short abstracts:
Experimental Section 1: In Vivo Imaging Sheds Light on the Susceptibility and Permis-sivity of Carassius auratus to CyHV-2 According to Developmental Stage. There has been a lack of comprehensive studies regarding CyHV-2 host susceptibility and permissivity across different develop-mental stages, and the primary portal of viral entry into the host remains unclear. To address these knowledge gaps, we engineered the first recombinant strain of CyHV-2 expressing bioluminescent and fluorescent reporter genes. Infection of goldfish hosts with this recombinant strain through immersion enabled us to utilize various in vivo imaging techniques to elucidate the spatiotemporal aspects of CyHV-2 replication during larval, juvenile, and adult developmental stages. Although larvae were less susceptible compared to later developmental stages, they exhibited the highest permissivity to CyHV-2 replication, resulting in rapid systemic infection and high mortality. Permissivity to CyHV-2 decreased as development progressed, with adults being the least permissive and consequently experiencing the lowest mortality rates. Throughout all developmental stages, the skin was identified as the most suscep-tible and permissive organ to infection during the early stages post-infection, suggesting that it serves as the primary portal of entry into these hosts. These findings collectively offer valuable fundamental insights into CyHV-2 pathogenesis and epidemiology in Carassius auratus, with significant relevance to other economically important virus-host models within the same genus.
Experimental section 2: Susceptibility and Permissivity of Zebrafish (Danio rerio) Larvae to Cypriniviruses. The zebrafish (Danio rerio) has emerged as a useful model organism in virology, facilitating the study of various viruses. Notably, it has proven to be valuable in studying a wide variety of viruses. Furthermore, it can easily be genetically manipulated, making it a valuable tool in studying virus-host interaction. In this study, a zebrafish model was tested to determine its susceptibility and permissivity to CyHV-2 relative to other viruses in the genus Cyprinivirus. Our investigation revealed that zebrafish larvae were not susceptible to these viruses when challenged by immersion. Nevertheless, infections with CyHV-2 and CyHV-3 could be established using artificial infection models both in vitro (utilizing zebrafish cell lines) and in vivo (via microinjection of larvae). However, these infections were transient, with rapid viral clearance correlated with apoptosis-like death of infected cells.
Experimental Section 3: In vitro and In vivo Characterization of novel CyHV-2 Strains from The Netherlands. 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 via challenge by immersion, particularly in the context of European host populations. These kinds of strains are much more compatible with experimental designs that are representative of natural infection. In this study, we isolated three novel strains of CyHV-2 originating from high mortal-ity outbreaks in The Netherlands, which we refer to as NL-1, NL-2, and NL-3. Genome sequencing and phylogenetic analyses revealed that these newly isolated strains are distinct from known strains and from each other. The three strains also exhibited significant differences in terms of in vitro growth ki-netics, 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 mor-tality of ~40%, while parallel experiments with the CyHV-2 reference strain ST-J1 resulted in no mor-tality. In summary, this study revealed that the NL-2 strain is (i) compatible with stable passaging in vitro, with acceptable replication kinetics in this environment, and (ii) highly virulent and capable of inducing CyHV-2 related mortality in adult Shubunkin goldfish when challenged via immersion. This resulted in the description of a new CyHV-2 in vivo infection model, much more compatible with experimental designs that are required to be representative of natural infection.