Biology subjects

Pichon, M.

Publications and source records attributed to Pichon, M..

2 recordsLinked to original sources

bioRxiv2018

Evolution of influenza genome diversity during infection in immunocompetent patients

IntroductionMinor frequency viruses play many important roles during viral infection that cannot be explained by the consensus sequence alone. In influenza, immunosuppressed individuals appear to generate numerous viral variants, leading to subpopulations with important role in infection. The objective of the present study was to describe viral diversification over time in immunocompetent patients during influenza virus infection.\n\nMethodsAll clinical records of patients admitted to the Lyon university hospital (Lyon, France) during the influenza infection epidemics of the 2010-2015 period and sampled at least twice during their clinical management were retrospectively analyzed. To estimate performance of the sequencing procedures, well-characterized plasmids containing each of the 8 segments of influenza viruses were used as quality controls. Diversity, i.e. the number of validated single nucleotide variants, was analyzed to compare characteristics over time and according to clinical severity (mild, severe with neurological complications, severe with respiratory complications).\n\nResultsAfter validation on quality controls (n=51), and verification of possible confusion bias, a 5%-threshold of detection was applied to clinical viral sequences (n=29). At this threshold, amino-acid coordinates (n=183/4,246, 4.31%) were identified as having at least one mutation during clinical course, independently of the clinical severity. Considering a threshold of 4 days of symptoms, as a limit for early and late sampling, diversity was significantly higher in late samples for the mild group, compared to both early mild and severe groups (p<0.05). At a single-segment scale, for PB2-coding segment, diversity was significantly higher in early samples of the neurological group than in both early and late samples in the respiratory group and for late samples in the mild group (p<0.05). For the NS1-coding segment, significant differences were observed between initial diversity of mild and severe patients, as for early and late samples in mild patients (p<0.01). Discussion. This study is the first describing diversity through time, associating biological and clinical information during viral diversification, during the infection of an immunocompetent human host. This latter opens a large field of investigation in infectious disease management using next-generation sequencing and suggest development of new therapies, focusing on non-antigenic viral properties, in non-vaccine fields of research

microbiology↗

bioRxiv2018

Quality control implementation for universal characterization of DNA and RNA viruses in clinical respiratory samples using single metagenomic next-generation sequencing workflow

BackgroundIn recent years, metagenomic Next-Generation Sequencing (mNGS) has increasingly been used for an accurate assumption-free virological diagnosis. However, the systematic workflow evaluation on clinical respiratory samples and implementation of quality controls (QCs) is still lacking.\n\nMethodsA total of 3 QCs were implemented and processed through the whole mNGS workflow: a notemplate-control to evaluate contamination issues during the process; an internal and an external QC to check the integrity of the reagents, equipment, the presence of inhibitors, and to allow the validation of results for each sample. The workflow was then evaluated on 37 clinical respiratory samples from patients with acute respiratory infections previously tested for a broad panel of viruses using semi-quantitative real-time PCR assays (28 positive samples including 6 multiple viral infections; 9 negative samples). Selected specimens included nasopharyngeal swabs (n = 20), aspirates (n = 10), or sputums (n = 7).\n\nResultsThe optimal spiking level of the internal QC was first determined in order to be sufficiently detected without overconsumption of sequencing reads. According to QC validation criteria, mNGS results were validated for 34/37 selected samples. For valid samples, viral genotypes were accurately determined for 36/36 viruses detected with PCR (viral genome coverage ranged from 0.6% to 100%, median = 67.7%). This mNGS workflow allowed the detection of DNA and RNA viruses up to a semi-quantitative PCR Ct value of 36. The six multiple viral infections involving 2 to 4 viruses were also fully characterized. A strong correlation between results of mNGS and real-time PCR was obtained for each type of viral genome (R2 ranged from 0.72 for linear single-stranded (ss) RNA viruses to 0.98 for linear ssDNA viruses).\n\nConclusionsAlthough the potential of mNGS technology is very promising, further evaluation studies are urgently needed for its routine clinical use within a reasonable timeframe. The approach described herein is crucial to bring standardization and to ensure the quality of the generated sequences in clinical setting. We provide an easy-to-use single protocol successfully evaluated for the characterization of a broad and representative panel of DNA and RNA respiratory viruses in various types of clinical samples.

microbiology↗