Characterization of Quasispecies of Pandemic 2009 Influenza A Virus (A/H1N1/2009) by De Novo Sequencing Using a Next-Generation DNA Sequencer

Kuroda, Makoto; Katano, Harutaka; Nakajima, N.; Tobiume, Minoru; Ainai, Akira; Sekizuka, Tsuyoshi; Hasegawa, Hideki; Tashiro, Masato; Sasaki, Yuko; Arakawa, Yoshichika; Hata, Satoru; Watanabe, Masahide; Sata, Tetsutaro

doi:10.1371/journal.pone.0010256

Cited by 106 publications

(94 citation statements)

References 30 publications

(39 reference statements)

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“…When the sequences were translated to amino acids, the substitution from aspartic acid (D) to glycine (G) (D222G) was a major clone, whereas the substitution from aspartic acid (D) to asparagine (N) (D222N) was a minor clone in the lung. This was consistent with the results of our previous analysis in frozen lung tissues using de novo sequencing with a next-generation DNA sequencer, 29 an effective analytical tool, which needs a high number of copies of viral RNA for analysis. In this study, using formalin-fixed and paraffin-embedded specimens, and a direct sequencing method, we also detected different 2009 H1N1 virus clones with different nucleic acids in the tracheal sample from case 1 (Figure 4), where the D222G substitution was a minor clone and 222D was a major clone.…”

Section: D222g Substitutions In the Hemagglutinin Of 2009 H1n1 Virussupporting

confidence: 92%

Histopathological and immunohistochemical findings of 20 autopsy cases with 2009 H1N1 virus infection

et al. 2012

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, were histopathologically analyzed. Hematoxylin-eosin staining, immunohistochemistry for type A influenza nucleoprotein antigen, and real-time reverse transcription-PCR assay for viral RNA were performed on formalin-fixed and paraffin-embedded specimens. In addition, the D222G amino acid substitution in influenza virus hemagglutinin, which binds to specific cell receptors, was analyzed in formalinfixed and paraffin-embedded trachea and lung sections by direct sequencing of PCR-amplified products. There were several histopathological patterns in the lung according to the most remarkable findings in each case: acute diffuse alveolar damage (DAD) with a hyaline membrane (four cases), organized DAD (one case), acute massive intra-alveolar edema with variable degrees of hemorrhage (three cases), neutrophilic bronchopneumonia (five cases) and tracheobronchitis with limited histopathological changes in alveoli (four cases). In two cases, the main findings were due to preexisting disease. Influenza virus antigen was only detected in the respiratory tract in 10 cases by immunohistochemistry. The antigen was detected in type II pneumocytes (three cases) in the epithelial cells of the trachea, bronchi and glands (six cases), and in the epithelial cells in both of the above (one case). The four cases with acute DAD presented with antigen-positive type II pneumocytes. In one case, the D222G substitution was detected in the lung as a major sequence, although 222D was prominent in the trachea, suggesting that selection of the viral clones occurred in the respiratory tract. In five

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Section: D222g Substitutions In the Hemagglutinin Of 2009 H1n1 Virussupporting

confidence: 92%

Histopathological and immunohistochemical findings of 20 autopsy cases with 2009 H1N1 virus infection

et al. 2012

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“…This approach has been previously used to resolve genomic sequences of influenza virus [6]; for example, Greninger et al reported that de novo assembled contigs had 90.3% coverage using 60bp short-reads [5], which is in accordance with our results (Table 2). Given the short length of the influenza genome and the structural simplicity of their genes, as compared with most mammal genes, the number of reads covering the target sequence is the most important factor for the success of this approach rather than the election of the The sequencing run generated 20x10 6 paired-end reads, 90bp.…”

Section: Discussionsupporting

confidence: 91%

“…Our results (Table 1) and also previously published studies [6,7] show that direct RNA sequencing can provide very high coverage of the viral genome; however, there can be clinical samples where the number of virus-matching reads is low [29] and pre-amplification is still an option that may need consideration. The selection of the sequencing platform will determine the number of reads that can be obtained.…”

Section: Discussionsupporting

confidence: 73%

“…Next-generation sequencing (NGS) allows the detection of pathogens when only little prior knowledge of their genomes is available and without the need for target-specific PCR primers. Additionally, NGS technologies deliver a large amount of genomic information that allows the study of additional aspects such as development of resistance to antivirals, variety of quasi-species and determinants of adaptation to different host species [6,7] .…”

Section: Introductionmentioning

confidence: 99%

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

Lin

Farooqui

et al. 2014

J Infect Dev Ctries

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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.

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“…The majority of A/H1N1pdm09 clones in the frozen lung tissue of case 2009 reportedly harbored the D222G mutation, which enables the virus to infect alveolar epithelial cells (11). We reexamined the HA gene sequence of the 10 A/H1N1pdm09 clones from the FFPE lung sections of case 2009 and noted that 9 of the 10 clones harbored the D222G mutation (Fig.…”

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confidence: 99%

Histopathological Findings of Lung with A/H1N1pdm09 Infection-Associated Acute Respiratory Distress Syndrome in the Post-Pandemic Season

et al. 2017

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SUMMARY:We herein report the pulmonary histopathological findings of an autopsy case of postpandemic season A/H1N1pdm09 infection-associated acute respiratory distress syndrome (ARDS). The lung histology predominantly exhibited findings indicative of the exudative phase of diffuse alveolar damage, with similar inflammation severity observed in all sections. Furthermore, the lung sections only showed a few A/H1N1pdm09 antigen-positive cells along with a low viral RNA copy number. The sequence of the viral hemagglutinin receptor binding site identified a preference for a-2,6 linked sialic acid, suggesting low alveolar epithelial cell infectivity. The pathological findings, in this case, differed in several aspects from those of the first autopsy case of A/H1N1pdm09 infection-associated ARDS in Japan, reported during the 2009 pandemic season. In conclusion, pathological and molecular biological examinations suggested that in the post-pandemic season A/H1N1pdm09 infection, the infectionassociated ARDS was not caused by direct infection-induced damage to the alveolar epithelial cells but was rather a result of indirect sepsis-mediated endothelial cell damage.

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Characterization of Quasispecies of Pandemic 2009 Influenza A Virus (A/H1N1/2009) by De Novo Sequencing Using a Next-Generation DNA Sequencer

Cited by 106 publications

References 30 publications

Histopathological and immunohistochemical findings of 20 autopsy cases with 2009 H1N1 virus infection

Histopathological and immunohistochemical findings of 20 autopsy cases with 2009 H1N1 virus infection

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

Histopathological Findings of Lung with A/H1N1pdm09 Infection-Associated Acute Respiratory Distress Syndrome in the Post-Pandemic Season

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