Susceptibility and permissivity of zebrafish (Danio rerio) larvae to cypriniviruses

The cyprinid herpesvirus 3 (CyHV-3), also known as koi herpesvirus (KHV), is a member of the genus Cyprinivirus, family Alloherpesviridae, order Herpesvirales. It is responsible of a lethal disease in common and koi carp (Cyprinus carpio), resulting in massive economic losses, and is now the most studied virus among fish herpesviruses. In addition to CyHV-3, the genus Cyprinivirus contains two other economically important viruses, namely the anguillid herpesvirus 1 (AngHV-1, affecting eel species) and cyprinid herpesvirus 2 (CyHV-2, affecting goldfish, Crucian carp, and Gibel carp). A recent study demonstrated that CyHV-3 induces an abortive infection after intraperitoneal inoculation of adult zebrafish (Danio rerio), in addition to being able to infect zebrafish cell lines. The zebrafish, a member of the family Cyprinidae, is an experimental model which has become increasingly used in virology, notably for studying infectious diseases affecting reared fish. The broad objectives of the present thesis were to compare AngHV-1, CyHV-2, and CyHV-3 in terms of their ability to infect zebrafish models both in vitro and in vivo and to investigate the potential of the zebrafish model to study the pathogenesis of these three cypriniviruses. Firstly, the susceptibility and permissivity of the ZF4 cell line and zebrafish larvae to infection with AngHV-1, CyHV-2, and CyHV-3, was investigated, using recombinant strains expressing green fluorescent proteins (GFP) and luciferase as reporter genes. Viral replication was monitored using time-lapse fluorescence microscopy, and bioluminescence imaging (IVIS). Our results showed that in vitro, cells were susceptible to infection with all viruses, but only permissive to CyHV-2 and CyHV-3, while in vivo, larvae were only susceptible to CyHV-2 and CyHV-3 following inoculation by microinjection. Furthermore, while susceptible, it was unclear to what extent larvae were permissive to CyHV-2 and CyHV-3. However, both in vitro and in vivo, infections established by all these viruses were ultimately cleared rapidly after infection, with live microscopy indicating the potential implication of programmed cell death in this response. Then, a transcriptomic analysis of CyHV-3-infected larvae was conducted in order to further characterize the response to CyHV-3 infection in this model. It revealed up-regulation of many known interferon-stimulated genes, in particular those encoding nucleic acid sensors, mediators of programmed cell death and related genes. Also, uncharacterized non-coding RNA genes and retrotransposons were among those most up-regulated. Interestingly, this retrotransposon re-activation/up-regulation in response to infection may be beneficial, as their cytoplasmic RNA and/or DNA genome intermediates may potentially act as ligands for PRRs, thus enhancing the innate immune response to viral infection. The transcriptomic analysis supported a potentially important role of protein kinase R (PKR) and a related protein kinase containing Z-DNA binding domains (PKZ) in the programmed cell death response. The impact of these kinases on CyHV-3 clearance was investigated, using CRISPR/Cas9-generated PKR-KO and PKZ-KO zebrafish mutants. The results of these experiments revealed that the knockout of PKR and PKZ did not impair the clearance of CyHV-3 infections in zebrafish larvae. We hypothesize that even at this early developmental stage, the immune system exhibits enough redundancy to mount a sufficient response in the absence of these genes. This is the first report of the generation and use of PKR and/or PKZ-KO zebrafish mutants, and they will represent useful subjects for further characterization and the study of other viruses in zebrafish models.
In conclusion, these findings show the importance of the innate immune response alone in clearing viral infection and emphasize the high degree of adaptation that cypriniviruses have undergone to facilitate successful persistence and transmission within their respective natural hosts. Finally, this project highlights the potential value of the CyHV-3-zebrafish model versus CyHV-3-carp models to study the fundamental features of virus-host interactions.