Abstract
Evolution of viruses is the result of a tradeoff between genetic diversification and selection. Though it has long been considered that single nucleotide polymorphisms (SNPs) were the main drivers of genetic diversity, it is now acknowledged that structural variations (SVs) may play a great role in virus diversity. The large dsDNA Cyprinid herpesvirus 3 (CyHV-3; 295 kbp) is one of the most pathogenic aquatic viruses. CyHV-3 mainly infects common carp and koi fish and causes mass mortalities, up to 100%. CyHV-3 exists in its host as a population of closely related genetic variants, or haplotypes. Knowledge on the mechanisms and evolutionary consequences of this intra-host diversity is essential in developing preventive strategies such as vaccines.An earlier study had revealed that the composition of haplotype assemblies may quickly vary along consecutive in vitro infectious cycles, resulting in quick phenotypic modifications. Of interest, a deletion of 1363 pb in the ORF150 (ORF150-del) was associated with a loss of virulence. These findings laid the basis of the present work, around three main objectives: 1) get more insights into the evolutionary mechanisms underlying such haplotype assemblies in cell culture, with a focus on ORF150; 2) investigate the prevalence of ORF150-del haplotype and other structural variations in this genomic region in vivo; 3) explore the accumulation pattern of SVs and SNPs in CyHV-3 genome under replicate cell cultures and after application of a thermal stress.In this work, long-read sequencing was applied for the first time on a Tawainese isolate of CyHV-3, which had been passaged 100 times on cell culture. Genome Analyses revealed that SVs were the major source of polymorphism. SVs displayed a wide distribution along CyHV-3 genome and exhibited high turnover dynamics, with a clear bias towards inversion and deletion events. Analysis of the ORF150 region in ten intermediate cell passages highlighted several deletions, inversions and insertions that deeply altered the structure of ORF150. Results suggested that SV turnovers and defective genomes represent key drivers in the viral population dynamics and in vitro evolution of CyHV-3.For the in vivo study, 236 fish from 43 different carp farms of Indonesia were screened for the presence of CyHV-3. Results confirmed a high prevalence of CyHV-3 (nearly 70%), both in symptomatic and asymptomatic common carp. However, in contrast with the results obtained in vitro, long read sequencing of the ORF150 region revealed a low level of genetic diversity and the absence of the 1363-pb deletion.To address the third objective, an Italian isolate was propagated in 10 identical subcultures and serially passaged 50 times under identical experimental conditions. A thermal stress was applied at passage 26 (P26). Analysis of genome-wide sequences revealed that SNPs accumulated steadily after successive passages, with a uniform distribution along the genome. The thermal stress had apparently no effect on their accumulation in the viral population. In contrast, the accrual of SVs was slower and was obviously affected by the thermal stress. Interestingly, CyHV-3 showed stronger and earlier cytopathic effects from P38 onwards. This observation was concomitant with the fixation in the viral genomes of all subcultures of a specific SNP in ORF131, which was previously demonstrated to be involved in an increased in vitro fitness of CyHV-3. Finally, no deletion in ORF150 could be detected, contrary to what could be expected from the first study.The results presented here suggest that SVs play a substantial role in the evolution of CyHV-3 at least in vitro, when environmental pressures are absent. They indicate that ORF150-del haplotype appeared randomly and highlight how fundamental research in virology may answer applied questions.