title: Discovery of a divergent HPIV4 from respiratory secretions using second and third generation metagenomic sequencing.
abstract: Molecular detection of viruses has been aided by high-throughput sequencing, permitting the genomic characterization of emerging strains. In this study, we comprehensively screened 500 respiratory secretions from children with upper and/or lower respiratory tract infections for viral pathogens. The viruses detected are described, including a divergent human parainfluenza virus type 4 from GS FLX pyrosequencing of 92 specimens. Complete full-genome characterization of the virus followed, using Single Molecule, Real-Time (SMRT) sequencing. Subsequent "primer walking" combined with Sanger sequencing validated the RS platform's utility in viral sequencing from complex clinical samples. Comparative genomics reveals the divergent strain clusters with the only completely sequenced HPIV4a subtype. However, it also exhibits various structural features present in one of the HPIV4b reference strains, opening questions regarding their lifecycle and evolutionary relationships among these viruses. Clinical data from patients infected with the strain, as well as viral prevalence estimates using real-time PCR, is also described.
authors.affiliation: Centre for GeoGenetics, Natural History Museum of Denmark, University of Copenhagen, Øster Voldgade 5-7, 1350 Copenhagen, Denmark.
authors.forename: David E
authors.initials: DE
authors.lastname: Alquezar-Planas
authors.forename: Tobias
authors.initials: T
authors.lastname: Mourier
authors.forename: Christian A W
authors.initials: CA
authors.lastname: Bruhn
authors.forename: Anders J
authors.initials: AJ
authors.lastname: Hansen
authors.forename: Sarah Nathalie
authors.initials: SN
authors.lastname: Vitcetz
authors.forename: Søren
authors.initials: S
authors.lastname: Mørk
authors.forename: Jan
authors.initials: J
authors.lastname: Gorodkin
authors.forename: Hanne Abel
authors.initials: HA
authors.lastname: Nielsen
authors.forename: Yan
authors.initials: Y
authors.lastname: Guo
authors.forename: Anand
authors.initials: A
authors.lastname: Sethuraman
authors.forename: Ellen E
authors.initials: EE
authors.lastname: Paxinos
authors.forename: Tongling
authors.initials: T
authors.lastname: Shan
authors.forename: Eric L
authors.initials: EL
authors.lastname: Delwart
authors.forename: Lars P
authors.initials: LP
authors.lastname: Nielsen
mesh_terms: D001483:Base Sequence; D014644:Genetic Variation; D016679:Genome, Viral; D059014:High-Throughput Nucleotide Sequencing; D006801:Humans; D056186:Metagenomics / Q000379:methods; D008969:Molecular Sequence Data; D016366:Open Reading Frames; D029482:Parainfluenza Virus 4, Human / Q000145:classification / Q000235:genetics* / Q000302:isolation & purification; D010802:Phylogeny; D015995:Prevalence; D012141:Respiratory Tract Infections / Q000453:epidemiology / Q000821:virology*; D016415:Sequence Alignment