title: Characterization of quasispecies of pandemic 2009 influenza A virus (A/H1N1/2009) by de novo sequencing using a next-generation DNA sequencer.
abstract: Pandemic 2009 influenza A virus (A/H1N1/2009) has emerged globally. In this study, we performed a comprehensive detection of potential pathogens by de novo sequencing using a next-generation DNA sequencer on total RNAs extracted from an autopsy lung of a patient who died of viral pneumonia with A/H1N1/2009. Among a total of 9.4x10(6) 40-mer short reads, more than 98% appeared to be human, while 0.85% were identified as A/H1N1/2009 (A/Nagano/RC1-L/2009(H1N1)). Suspected bacterial reads such as Streptococcus pneumoniae and other oral bacteria flora were very low at 0.005%, and a significant bacterial infection was not histologically observed. De novo assembly and read mapping analysis of A/Nagano/RC1-L/2009(H1N1) showed more than x200 coverage on average, and revealed nucleotide heterogeneity on hemagglutinin as quasispecies, specifically at two amino acids (Gly(172)Glu and Gly(239)Asn of HA) located on the Sa and Ca2 antigenic sites, respectively. Gly239 and Asn239 on antigenic site Ca2 appeared to be minor amino acids compared with the highly distributed Asp239 in H1N1 HAs. This study demonstrated that de novo sequencing can comprehensively detect pathogens, and such in-depth investigation facilitates the identification of influenza A viral heterogeneity. To better characterize the A/H1N1/2009 virus, unbiased comprehensive techniques will be indispensable for the primary investigations of emerging infectious diseases.
authors.affiliation: Laboratory of Bacterial Genomics, Pathogen Genomics Center, National Institute of Infectious Diseases, Tokyo, Japan. makokuro@nih.go.jp
authors.forename: Makoto
authors.initials: M
authors.lastname: Kuroda
authors.forename: Harutaka
authors.initials: H
authors.lastname: Katano
authors.forename: Noriko
authors.initials: N
authors.lastname: Nakajima
authors.forename: Minoru
authors.initials: M
authors.lastname: Tobiume
authors.forename: Akira
authors.initials: A
authors.lastname: Ainai
authors.forename: Tsuyoshi
authors.initials: T
authors.lastname: Sekizuka
authors.forename: Hideki
authors.initials: H
authors.lastname: Hasegawa
authors.forename: Masato
authors.initials: M
authors.lastname: Tashiro
authors.forename: Yuko
authors.initials: Y
authors.lastname: Sasaki
authors.forename: Yoshichika
authors.initials: Y
authors.lastname: Arakawa
authors.forename: Satoru
authors.initials: S
authors.lastname: Hata
authors.forename: Masahide
authors.initials: M
authors.lastname: Watanabe
authors.forename: Tetsutaro
authors.initials: T
authors.lastname: Sata
mesh_terms: D014644:Genetic Variation; D019267:Hemagglutinin Glycoproteins, Influenza Virus; D006801:Humans; D053118:Influenza A Virus, H1N1 Subtype / Q000235:genetics / Q000302:isolation & purification*; D008168:Lung / Q000821:virology; D058873:Pandemics; D011024:Pneumonia, Viral / Q000821:virology; D012313:RNA / Q000302:isolation & purification; D012367:RNA, Viral / Q000032:analysis; D017422:Sequence Analysis, DNA / Q000295:instrumentation*
chemical_list: D019267:Hemagglutinin Glycoproteins, Influenza Virus; D012367:RNA, Viral; D012313:RNA