The anthropogenic spread of pathogens contributes to the decline of some species. However, restocking programs implemented to counteract the decline of such populations can also contribute to the spread of pathogens, in particular for endangered species that cannot be bred in captivity, where populations used in restocking must be sourced from the wild with no control over sanitary status. A prime example of this is the endangered species European eel (Anguilla anguilla). As part of eel restocking programs, glass eels are captured in estuaries for later release further inland. In cases where release occurs immediately after capture, pre-release quarantine is important for mitigation of sanitary risks. In this study, we explored the impact of different quarantine conditions on glass eel sanitary status, morphology, behaviour and quarantine/post-quarantine survival in order to evaluate the feasibility and optimization of quarantine stages. Replicate experiments were conducted in 2017 and 2018, using glass eels of different geographical origin. The results suggest that glass eels entering estuaries are free of pathogens and so that their capture at this early stage reduces the risk of the anthropogenic introduction of pathogens through restocking programs. They support the use of a 15-day pre-release quarantine period to allow easier management of release processes and the implementation of prophylactic and therapeutic measures as part of these useful conservation programs.

Anguillid herpesvirus 1 (AngHV-1) is a pathogen of eels and a member of the genus Cyprinivirus in the family Alloherpesviridae. We have compared the biological and genomic features of different AngHV-1 strains, focusing on their growth kinetics in vitro and genetic content, diversity, and recombination. Comparisons based on three core genes conserved among alloherpesviruses revealed that AngHV-1 exhibits a slower rate of change and less positive selection than other cypriniviruses. We propose that this may be linked to major differences in host species and corresponding epidemiological circumstances. Efforts to derive evolutionary rate estimates for cypriniviruses under various theoretical models were ultimately unrewarding. We highlight the potential value of future collaborative efforts towards generating short-term evolutionary rate estimates based on known sequence sampling dates. Finally, we revealed that there is significantly less genetic diversity in core gene sequences within cyprinivirus species clades compared to species in the family Herpesviridae. This suggests that cyprinivirus species may have undergone much more vigorous purifying selection post species clade divergence. We discuss whether this may be linked to biological and anthropogenic factors or to sampling bias, and we propose that the comparison of short-term evolutionary rates between species may provide further insights into these differences.

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The zebrafish (Danio rerio) represents an increasingly important model organism in virology. We evaluated its utility in the study of economically important viruses from the genus Cyprinivirus (anguillid herpesvirus 1, cyprinid herpesvirus 2 and cyprinid herpesvirus 3 (CyHV-3)). This revealed that zebrafish larvae were not susceptible to these viruses after immersion in contaminated water, but that infections could be established using artificial infection models in vitro (zebrafish cell lines) and in vivo (microinjection of larvae). However, infections were transient, with rapid viral clearance associated with apoptosis-like death of infected cells. Transcriptomic analysis of CyHV-3-infected larvae revealed upregulation of interferon-stimulated genes, in particular those encoding nucleic acid sensors, mediators of programmed cell death and related genes. It was notable that uncharacterized non-coding RNA genes and retrotransposons were also among those most upregulated. CRISPR/Cas9 knockout of the zebrafish gene encoding protein kinase R (PKR) and a related gene encoding a protein kinase containing Z-DNA binding domains (PKZ) had no impact on CyHV-3 clearance in larvae. Our study strongly supports the importance of innate immunity-virus interactions in the adaptation of cypriniviruses to their natural hosts. It also highlights the potential of the CyHV-3-zebrafish model, versus the CyHV-3-carp model, for study of these interactions.