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

Wong, T. H., Dearlove, B. L., Hedge, J., Giess, A. P., Piazza, P., Trebes, A., Paul, J., Smit, E., Smith, E. G., Sutton, J. K., Wilcox, M. H., Dingle, K. E., Peto, T. E., Crook, D. W., Wilson, D. J. and D. H. Wyllie (2013)
Virology Journal 10: 335 (pdf)

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 [pound sign]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 <0.003%. Intra-host nucleotide sequence diversity was rare, but detectable by mapping short sequence reads onto its de novo assembled consensus. Genomes similar to the Sydney 2012 strain caused 78% (18/23) of cases, consistent with its previously documented association with the winter 2012 global outbreak. Interestingly, phylogenetic analysis and recombination detection analysis of the consensus sequences identified two related viruses as recombinants, containing sequences in prior circulation to Sydney 2012 in open reading frame (ORF) 2.

Conclusion

Our approach facilitates the rapid determination of complete norovirus genomes. This method provides high resolution of full norovirus genomes which, when coupled with detailed epidemiology, may improve the understanding of evolution and control of this important healthcare-associated pathogen.