title: Random amplification and pyrosequencing for identification of novel viral genome sequences.
abstract: ssRNA viruses have high levels of genomic divergence, which can lead to difficulty in genomic characterization of new viruses using traditional PCR amplification and sequencing methods. In this study, random reverse transcription, anchored random PCR amplification, and high-throughput pyrosequencing were used to identify orthobunyavirus sequences from total RNA extracted from viral cultures of acute febrile illness specimens. Draft genome sequence for the orthobunyavirus L segment was assembled and sequentially extended using de novo assembly contigs from pyrosequencing reads and orthobunyavirus sequences in GenBank as guidance. Accuracy and continuous coverage were achieved by mapping all reads to the L segment draft sequence. Subsequently, RT-PCR and Sanger sequencing were used to complete the genome sequence. The complete L segment was found to be 6936 bases in length, encoding a 2248-aa putative RNA polymerase. The identified L segment was distinct from previously published South American orthobunyaviruses, sharing 63% and 54% identity at the nucleotide and amino acid level, respectively, with the complete Oropouche virus L segment and 73% and 81% identity at the nucleotide and amino acid level, respectively, with a partial Caraparu virus L segment. The result demonstrated the effectiveness of a sequence-independent amplification and next-generation sequencing approach for obtaining complete viral genomes from total nucleic acid extracts and its use in pathogen discovery.
authors.affiliation: Viral Diseases Branch, Walter Reed Army Institute of Research, Silver Spring, Maryland 20910, USA. jun.hang.ctr@us.army.mil
authors.forename: Jun
authors.initials: J
authors.lastname: Hang
authors.forename: Brett M
authors.initials: BM
authors.lastname: Forshey
authors.forename: Tadeusz J
authors.initials: TJ
authors.lastname: Kochel
authors.forename: Tao
authors.initials: T
authors.lastname: Li
authors.forename: Víctor Fiestas
authors.initials: VF
authors.lastname: Solórzano
authors.forename: Eric S
authors.initials: ES
authors.lastname: Halsey
authors.forename: Robert A
authors.initials: RA
authors.lastname: Kuschner
mesh_terms: D000293:Adolescent; D000330:Aedes; D000818:Animals; D002522:Chlorocebus aethiops; D020451:Contig Mapping; D017931:DNA Primers / Q000235:genetics; D057225:Data Mining; D005260:Female; D019084:Fluorescent Antibody Technique, Indirect; D016679:Genome, Viral; D006801:Humans; D008875:Middle Aged; D008969:Molecular Sequence Data; D058889:Molecular Typing; D016851:Orthobunyavirus / Q000145:classification / Q000235:genetics*; D012367:RNA, Viral / Q000235:genetics / Q000302:isolation & purification; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*; D017422:Sequence Analysis, DNA; D014709:Vero Cells; D014776:Virus Cultivation
chemical_list: D017931:DNA Primers; D012367:RNA, Viral