Next-generation sequencing and bioinformatic approaches to detect and analyze influenza virus in ferrets

Abstract

Introduction: Conventional methods used to detect and characterize influenza viruses in biological samples face multiple challenges due to the diversity of subtypes and high dissimilarity of emerging strains. Next-generation sequencing (NGS) is a powerful technique that can facilitate the detection and characterization of influenza, however, the sequencing strategy and the procedures of data analysis possess different aspects that require careful consideration. Methodology: The RNA from the lungs of ferrets infected with influenza A/California/07/2009 was analyzed by next-generation sequencing (NGS) without using specific PCR amplification of the viral sequences. Several bioinformatic approaches were used to resolve the viral genes and detect viral quasispecies. Results: The genomic sequences of influenza virus were characterized to a high level of detail when analyzing the short-reads with either the fast aligner Bowtie2, the general purpose aligner BLASTn or de novo assembly with Abyss. Moreover, when using distant viral sequences as reference, these methods were still able to resolve the viral sequences of a biological sample. Finally, direct sequencing of RNA samples did not provide sufficient coverage of the viral genome to study viral quasispecies, and, therefore, prior amplification of the viral segments by PCR would be required to perform this type of analysis. Conclusions: the introduction of NGS for virus research allows routine full characterization of viral isolates; however, careful design of the sequencing strategy and the procedures for data analysis are still of critical importance.

Ferreting Out Influenza Virus Pathogenicity and Transmissibility: Past and Future Risk Assessments in the Ferret Model

“…Molecular tagging of influenza viruses has provided insight into selection of quasispecies within an infected host and bottleneck dynamics during transmission to susceptible contacts (Varble et al 2014;Frise et al 2016), identifying differences in transmission dynamics between contact and respiratory droplet models. Similarly, next-generation sequencing of samples at both the within-host and between-host scales has provided an increased understanding of the role minor variant populations can contribute to these dynamics (Lin et al 2014;Varble et al 2014). As our molecular toolkit improves, so will the ability of virus-infected ferrets to provide insight into the dynamics of human influenza virus infections.…”

Metagenomic analysis of viral genetic diversity in respiratory samples from children with severe acute respiratory infection in China

“…In recent years, high-throughput next-generation sequencing (NGS) technology has become a powerful tool in pathogen detection compared with traditional methods, because it allows for the detection of pathogens without any advance genetic information. To date, NGS has been widely used for the characterization of community viral infections and novel pathogen identification in numerous hosts, including humans [4,5], pigs [6], ferrets [7], bats [8,9], cats [10] and rats [11]; it has also been used in locations such as vineyards [12] and in freshwater [13].…”

Genetic diversity of the 2013–14 human isolates of influenza H7N9 in China

“…Sequencing libraries were prepared by using the Nextera XT DNA Sample Preparation kit (Illumina) according to the manufacturer’s protocol, and 150 bp paired-end sequencing was performed in a MiSeq instrument (Illumina). The resulting short reads were aligned with respect to the sequences of A/Shanghai/1/2013 (H7N9) by Bowtie2 (2.2.3) [ 12 ] and the consensus sequences were generated with SAMtools (1.0) [ 13 ], as previously described [ 14 ]. The sequence variants were called with VarScan (2.3.6) [ 15 ] and only considered if causing aminoacid substitution and with a frequency ≥2%.…”