Genomic and biological comparisons of Anguillid herpesvirus 1 strains

The Anguillid herpesvirus 1 (AngHV-1), also known as herpesvirus anguillae (HVA), is a double stranded DNA virus that belongs to the Cyprinivirus genus of the Alloherpesviridae family. AngHV-1 is the etiologic agent of a lethal disease that affects many eel species, including European eel (Anguilla anguilla), Japanese eels (Anguilla japonica), American eels (Anguilla rostrata), Short finned eels (Anguilla australis), and Giant mottled eels (Anguilla marmorata), causing economic losses to the aquaculture sector. AngHV-1 is also thought to contribute to the drastic decline of eels in the wild. To date, the genome sequence of only two AngHV-1 isolates has been published and there is a limited knowledge on the pathogenesis of this viral species.
The work performed during this PhD is structured around two chapters:
1/ Genomic and in vitro biologic comparisons of AngHV-1 strains
The full-length genome of seven AngHV-1 strains obtained from different geographical origins were sequenced. Genomic and phylogenetic analyses of these 7 strains together with the two previously published sequences revealed a low genomic diversity among strains, although two genetic lineages could be identified. Recombination detection analyses indicated the occurrence of 5 recombination events, including some that occurred between the two distant lineages and one that occurred between strains of different geographic origin. Recombination analyses also suggested the existence of a third, yet unidentified lineage. The identification of disruptions within genes from some AngHV-1 strains allowed us to classify some as non-essential for viral growth in vitro. In vitro biological comparison of the seven sequenced AngHV-1 strains revealed differences of growth kinetics in cell culture up to 10 fold between the strains. This part of the work has been published in ‘Microorganisms’. I am the second co-first authors of this publication.
2/ In vivo biological comparisons of AngHV-1 study of AngHV-1 pathogenesis
To further compare strains in vivo, 4 representative viral strains were selected. For each selected strain, we produced a recombinant strain expressing a fusion reporter protein consisting of luciferase and copepod green fluorescent protein (LucGFP). Analysis of AngHV-1 LucGFP strains showed that the insertion of the cassette did not affect the expression of flanking genes (ORF32 and ORF33) or the replication of the virus in vitro. Next, we investigated the sensitivity of glass eels to AngHV-1 infection after inoculation of the LucGFP recombinant strains using an In vivo Imaging System (IVIS). IVIS analyses revealed that glass eels are resistant to AngHV-1 after infection by natural routes (oral contamination or immersion in contaminated water) but permissive to the infection after intraperitoneal inoculation. To compare the virulence of LucGFP strains and their parental wild type strains, glass eels were infected by intraperitoneal (IP) injection. The results revealed comparable and low virulence with an associated mortality below 10% for all strains tested. The recombinant strains produced were used to establish the developmental stage(s) of European eels that are the most sensitive to infection and to study the pathogenesis of AngHV-1 in sensitive subjects. I will be the second author of this publication using the recombinants that I produced and characterized.