Rôle des variations structurales dans la diversité génétique et l'évolution du Cyprinid herpesvirus 3 - TEL - Thèses en ligne
Thèse Année : 2023

Role of structural variations in the genetic diversity and evolution of Cyprinid herpesvirus 3

Rôle des variations structurales dans la diversité génétique et l'évolution du Cyprinid herpesvirus 3

Résumé

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.
The evolution of viruses is the result of a trade-off between genetic diversification and natural selection. Although point mutations (SNPs) have long been considered the main drivers of genetic diversity, it is now accepted that structural variations (SVRs) can play an important role in virus diversity. Cyprinid herpesvirus 3 (CyHV-3) is one of the most pathogenic aquatic viruses. It mainly infects common carp and koi carp, and causes mortalities of up to 100%. CyHV-3 exists in its host as a population of genetically related variants, or haplotypes.A previous study found that the composition of haplotype assemblies can rapidly change during in vitro serial passages, resulting in rapid phenotypic changes. In particular, a 1363 bp deletion in ORF150 (ORF150-del) was associated with a loss of virulence. These results laid the foundation for the present work, which is structured around three main objectives: 1) to better understand the evolutionary mechanisms underlying these haplotype assemblies in cell culture, with a particular interest in ORF150; 2) study the prevalence of ORF150-del deletion in vivo; 3) to explore the dynamics of ESR and SNP accumulation in the CyHV-3 genome in cell culture replicas and after the application of heat stress.Long fragment sequencing was performed for the first time on a Taiwanese isolate of CyHV-3, previously passed 100 times on cell culture. Sequencing results revealed that ESR was the main source of polymorphism. ESRs were evenly distributed across the viral genome and exhibited high turnover dynamics. Analysis of the ORF150 region during ten intermediate passes revealed several deletions, inversions and insertions. The results suggest that ESR and the presence of defective genomes represent key factors in the in vitro evolution of CyHV-3.For the in vivo study, 236 fish from 43 carp farms in Indonesia were examined for the presence of CyHV-3. The results confirmed a high prevalence of CyHV-3 (nearly 70%) in both symptomatic and asymptomatic fish. However, unlike the results obtained in vitro, the sequencingLong fragments of the ORF150 region revealed a low level of genetic diversity and the absence of the 1363 bp deletion.To address the third objective, an Italian isolate of CyHV-3 was amplified in parallel in 10 identical subcultures and then serially passed 50 times under identical experimental conditions. Heat stress was applied to passage 26 (P26). Analysis of the genomic sequences revealed that SNPs accumulated in a regular manner over successive passages, with a uniform distribution along the genome. Heat stress apparently had no effect on their accumulation in the viral population. In contrast, ESR accumulation was slower and clearly affected by heat stress. Cytopathic effects related to infection were found to be more severe and earlier from the 38th pass onwards. This observation coincided with the appearance of a specific SNP in ORF131, which a recent study showed was involved in increasing the virulence of CyHV-3 in vitro. Finally, no deletions in ORF150 could be detected, contrary to what could be expected from the first study.The results presented here suggest that ESRs play a major role in the evolution of CyHV-3 at least in vitro, in the absence of environmental pressures. They indicate that the haplotype ORF150-del arose randomly and highlight the interest of basic research in virology to answer applied questions.
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Dates et versions

tel-04867717 , version 1 (06-01-2025)

Identifiants

  • HAL Id : tel-04867717 , version 1

Citer

Nurul Fuandila. Rôle des variations structurales dans la diversité génétique et l'évolution du Cyprinid herpesvirus 3. Biodiversité et Ecologie. Université de Montpellier, 2023. Français. ⟨NNT : 2023UMONG061⟩. ⟨tel-04867717⟩
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