Contribution of Next-Generation Sequencing to Aquatic and Fish Virology

International audienceThe recent technological advances in nucleic acid sequencing, called next-generation sequencing (NGS), have revolutionized the field of genomics and have also influenced viral research. Aquatic viruses, and especially those infecting fish, have also greatly benefited from NGS technologies, which provide a huge amount of molecular information at a low cost in a relatively short period of time. Here, we review the use of the current high-throughput sequencing platforms with a special focus on the associated challenges (regarding sample preparation and bioinformatics) in their applications to the field of aquatic virology, especially for: (i) discovering novel viruses that may be associated with fish mortalities, (ii) elucidating the mechanisms of pathogenesis, and finally (iii) studying the molecular epidemiology of these pathogens

Improved assembly procedure of viral RNA genomes amplified with Phi29 polymerase from new generation sequencing data

International audienceNew sequencing technologies have opened the way to the discovery and the characterization of pathogenic viruses in clinical samples. However, the use of these new methods can require an amplification of viral RNA prior to the sequencing. Among all the available methods, the procedure based on the use of Phi29 polymerase produces a huge amount of amplified DNA. However, its major disadvantage is to generate a large number of chimeric sequences which can affect the assembly step. The pre-process method proposed in this study strongly limits the negative impact of chimeric reads in order to obtain the full-length of viral genomes

Improved assembly procedure of viral RNA genomes amplified with Phi29 polymerase from new generation sequencing data

BACKGROUND: New sequencing technologies have opened the way to the discovery and the characterization of pathogenic viruses in clinical samples. However, the use of these new methods can require an amplification of viral RNA prior to the sequencing. Among all the available methods, the procedure based on the use of Phi29 polymerase produces a huge amount of amplified DNA. However, its major disadvantage is to generate a large number of chimeric sequences which can affect the assembly step. The pre-process method proposed in this study strongly limits the negative impact of chimeric reads in order to obtain the full-length of viral genomes. FINDINGS: Three different assembly softwares (ABySS, Ray and SPAdes) were tested for their ability to correctly assemble the full-length of viral genomes. Although in all cases, our pre-processed method improved genome assembly, only its combination with the use of SPAdes allowed us to obtain the full-length of the viral genomes tested in one contig. CONCLUSIONS: The proposed pipeline is able to overcome drawbacks due to the generation of chimeric reads during the amplification of viral RNA which considerably improves the assembling of full-length viral genomes

Viral Exploration of Negative Acute Febrile Cases Observed during Chikungunya Outbreaks in Gabon

International audienceNon-malarial febrile illness outbreaks were documented in 2007 and 2010 in Gabon. After investigation, these outbreaks were attributed to the chikungunya and dengue viruses (CHIKV and DENV). However, for more than half of the samples analyzed, the causative agent was not identified. Given the geographical and ecological position of Gabon, where there is a great animal and microbial diversity, the circulation of other emerging viruses was suspected in these samples lacking aetiology. A total of 436 undiagnosed samples, collected between 2007 and 2013, and originating from 14 urban, suburban, and rural Gabonese locations were selected. These samples were used for viral isolation on newborn mice and VERO cells. In samples with signs of viral replication, cell supernatants and brain suspensions were used to extract nucleic acids and perform real-time RT-PCR targeting specific arboviruses, i.e., CHIKV, DENV, yellow fever, Rift Valley fever, and West Nile and Zika viruses. Virus isolation was conclusive for 43 samples either on newborn mice or by cell culture. Virus identification by RT-PCR led to the identification of CHIKV in 37 isolates. A total of 18 complete genomes and 19 partial sequences containing the E2 and E1 genes of CHIKV were sequenced using next-generation sequencing technology or the Sanger method. Phylogenetic analysis of the complete genomes showed that all the sequences belong to the East Central South Africa lineage. Furthermore, we identified 2 distinct clusters. The first cluster was made up of sequences from the western part of Gabon, whereas the second cluster was made up of sequences from the southern regions, reflecting the way CHIKV spread across the country following its initial introduction in 2007. Similar results were obtained when analyzing the CHIKV genes of the E2 and E1 structural proteins. Moreover, study of the mutations found in the E2 and E1 structural proteins revealed the presence of several mutations that facilitate the adaptation to the Aedes albopictus mosquito, such as E2 I211T and E1 A226V, in all the Gabonese CHIKV strains. Finally, sequencing of 6 additional viral isolates failed to lead to any conclusive identification

Genome-wide profiling of human papillomavirus DNA integration in liquid-based cytology specimens from a Gabonese female population using HPV capture technology

Abstract Human papillomavirus (HPV) is recognised as the cause of precancerous and cancerous cervical lesions. Furthermore, in high-grade lesions, HPV is frequently integrated in the host cell genome and associated with the partial or complete loss of the E1 and E2 genes, which regulate the activity of viral oncoproteins E6 and E7. In this study, using a double-capture system followed by high-throughput sequencing, we determined the HPV integration status present in liquid-based cervical smears in an urban Gabonese population. The main inclusion criteria were based on cytological grade and the detection of the HPV16 genotype using molecular assays. The rate of HPV integration in the host genome varied with cytological grade: 85.7% (6/7), 71.4% (5/7), 66.7% (2/3) 60% (3/5) and 30.8% (4/13) for carcinomas, HSIL, ASCH, LSIL and ASCUS, respectively. For high cytological grades (carcinomas and HSIL), genotypes HPV16 and 18 represented 92.9% of the samples (13/14). The integrated form of HPV16 genotype was mainly found in high-grade lesions in 71.4% of samples regardless of cytological grade. Minority genotypes (HPV33, 51, 58 and 59) were found in LSIL samples, except HPV59, which was identified in one HSIL sample. Among all the HPV genotypes identified after double capture, 10 genotypes (HPV30, 35, 39, 44, 45, 53, 56, 59, 74 and 82) were detected only in episomal form. Our study revealed that the degree of HPV integration varies with cervical cytological grade. The integration event might be a potential clinical prognostic biomarker for the prediction of the progression of neoplastic lesions

Nanopore sequencing of a monkeypox virus strain isolated from a pustular lesion in the Central African Republic

International audienceMonkeypox is an emerging and neglected zoonotic disease whose number of reported cases has been gradually increasing in Central Africa since 1980. This disease is caused by the monkeypox virus (MPXV), which belongs to the genus Orthopoxvirus in the family Poxviridae . Obtaining molecular data is particularly useful for establishing the relationships between the viral strains involved in outbreaks in countries affected by this disease. In this study, we evaluated the use of the MinION real-time sequencer as well as different polishing tools on MinION-sequenced genome for sequencing the MPXV genome originating from a pustular lesion in the context of an epidemic in a remote area of the Central African Republic. The reads corresponding to the MPXV genome were identified using two taxonomic classifiers, Kraken2 and Kaiju. Assembly of these reads led to a complete sequence of 196,956 bases, which is 6322 bases longer than the sequence previously obtained with Illumina sequencing from the same sample. The comparison of the two sequences showed mainly indels at the homopolymeric regions. However, the combined use of Canu with specific polishing tools such as Medaka and Homopolish was the best combination that reduced their numbers without adding mismatches. Although MinION sequencing is known to introduce a number of characteristic errors compared to Illumina sequencing, the new polishing tools allow a better-quality MinION-sequenced genome, thus to be used to help determine strain origin through phylogenetic analysis