Cyprinid herpesvirus 3 (CyHV-3), also known as koi herpesvirus (KHV), is a member of the genus Cyprinivirus, family Alloherpesviridae, order Herpesvirales. CyHV-3 is responsible of a lethal disease in common and koi carp (Cyprinus carpio) which are economically important fish species. Over the last two decades, several CyHV-3 strains have been isolated in different countries. Complete genome sequences of 4 strains (KHV-U, KHV-J, KHV-I and KHV GZ11) were available in the Genbank at the beginning of this PhD project, but no comparisons of the biologic properties of these strains have been reported. In the first part of the thesis, we have sequenced the genomes of a further seven strains from various geographical sources, and have compared their growth in vitro and virulence in vivo. This part illustrates the importance of coupling genomic and biologic comparisons of viral strains in order to enhance understanding of viral evolution and pathogenesis. It revealed a negative correlation among strains between viral growth in vitro and virulence in vivo. This observation suggested the existence of alleles conferring adaptation to cell culture but reduced fitness in vivo and vice versa. The objective of the second part of this thesis was to identify such alleles. We identified the formation of syncytium in vitro as a common trait of cell culture adapted CyHV-3 strains. Comparison of virion transmembrane proteins encoded by CyHV 3 strains and production of various recombinants demonstrated that the genetic determinism of this trait is linked to a single nucleotide polymorphism (SNP) of ORF131 encoding a structural glycoprotein. Recombinant strains differing only by this SNP (183A and 183T) were compared for their fitness in vitro and in vivo. The ORF131 183A allele associated with syncytium formation revealed the highest fitness in vitro but the lowest in vivo. Interestingly, co-infection experiments by the two recombinants encoding ORF131 SNP revealed both in vitro and in vivo that the variant with the highest fitness contributed to the purifying selection of the less fit variant through the inhibition of superinfection, which could contribute the low diversity observed in CyHV-3 species.