Whole genome sequencing and de novo assembly identifies Sydney-like variant noroviruses and recombinants during the winter 2012/2013 outbreak in England

• Published in: Virology journal 2013-11, Vol.10 (1), p.335-335, Article 335
• Main Authors: Wong, TH Nicholas, Dearlove, Bethany L, Hedge, Jessica, Giess, Adam P, Piazza, Paolo, Trebes, Amy, Paul, John, Smit, Erasmus, Smith, E Grace, Sutton, Julian K, Wilcox, Mark H, Dingle, Kate E, Peto, Tim E A, Crook, Derrick W, Wilson, Daniel J, Wyllie, David H
• Format: Article
• Language: English
• Subjects: Analysis; Biomedical research; Caliciviridae Infections - epidemiology; Caliciviridae Infections - virology; Care and treatment; Cluster Analysis; Diagnosis; Disease Outbreaks; DNA sequencing; England - epidemiology; Epidemiology; Evolution; Gastroenteritis; genome; Genome, Viral; Genomes; Health aspects; Health care reform; hospitals; host specificity; Humans; immunity; Medical care, Cost of; Medical research; Molecular Sequence Data; Norovirus; Norovirus - classification; Norovirus - genetics; Norovirus - isolation & purification; Nucleotide sequencing; open reading frames; pathogens; people; Phylogeny; Public health; quantitative polymerase chain reaction; Regulatory approval; Risk factors; RNA; RNA sequencing; RNA, Viral - genetics; sequence analysis; Sequence Analysis, DNA; Sequence Homology; Studies; Teaching hospitals; vaccine development; Viruses; Winter
• ISSN: 1743-422X; 1743-422X
• DOI: 10.1186/1743-422X-10-335

BACKGROUND: Norovirus is the commonest cause of epidemic gastroenteritis among people of all ages. Outbreaks frequently occur in hospitals and the community, costing the UK an estimated £110 m per annum. An evolutionary explanation for periodic increases in norovirus cases, despite some host-specific post immunity is currently limited to the identification of obvious recombinants. Our understanding could be significantly enhanced by full length genome sequences for large numbers of intensively sampled viruses, which would also assist control and vaccine design. Our objective is to develop rapid, high-throughput, end-to-end methods yielding complete norovirus genome sequences. We apply these methods to recent English outbreaks, placing them in the wider context of the international norovirus epidemic of winter 2012. METHOD: Norovirus sequences were generated from 28 unique clinical samples by Illumina RNA sequencing (RNA-Seq) of total faecal RNA. A range of de novo sequence assemblers were attempted. The best assembler was identified by validation against three replicate samples and two norovirus qPCR negative samples, together with an additional 20 sequences determined by PCR and fractional capillary sequencing. Phylogenetic methods were used to reconstruct evolutionary relationships from the whole genome sequences. RESULTS: Full length norovirus genomes were generated from 23/28 samples. 5/28 partial norovirus genomes were associated with low viral copy numbers. The de novo assembled sequences differed from sequences determined by capillary sequencing by