Abstract
Norovirus (NoV) is the most common cause of non-bacterial gastroenteritis and is a major agent associated with outbreaks of gastroenteritis. Conventional molecular genotyping analysis of NoV, used for the identification of transmission routes, relies on standard typing methods (STM) by Sanger-sequencing of only a limited part of the NoV genome, which could lead to wrong conclusions. Here, we combined a NoV capture method with next generation sequencing (NGS), which increased the proportion of norovirus reads by ~40 fold compared to NGS without prior capture. Of 15 NoV samples from 6 single-genotype outbreaks, near full-genome coverage (>90%) was obtained from 9 samples. Fourteen polymerase (RdRp) and 15 capsid (cap) genotypes were identified compared to 12 and 13 for the STM, respectively. Analysis of 9 samples from two mixed-genotype outbreaks identified 6 RdRp and 6 cap genotypes (two at >90% NoV genome coverage) compared to 4 and 2 for the STM, respectively. Furthermore, complete or partial sequences from the P2 hypervariable region were obtained from 7 of 8 outbreaks and a new NoV recombinant was identified. This approach could therefore strengthen outbreak investigations and could be applied to other important viruses in stool samples such as hepatitis A and enterovirus.
Highlights
Norovirus (NoV) is a positive-sense single-stranded RNA virus in the Caliciviridae family, and at least 40 genotypes divided into seven genogroups have been identified[1,2,3]
Despite a large sequencing depth allocated to each sample (1.5 to 5.5 million reads), only a relatively small proportion of the obtained reads were of NoV origin
Using the next generation sequencing (NGS) approach, complete and one partial genotype were detected in samples from 6 of the 8 outbreaks containing a single NoV genotype compared with 10 complete and five partial genotypes detected with the standard typing methods (STM) approach
Summary
Norovirus (NoV) is a positive-sense single-stranded RNA virus in the Caliciviridae family, and at least 40 genotypes divided into seven genogroups have been identified[1,2,3]. Due to insufficient reads mapping to the GI.Pb_GI.[6] reference in sample Ob-4-1, a valid phylogenetic comparison could not be performed, a BLASTN of the consensus sequence generated from the 22 mapped reads indicated this Phylogenetic analysis was performed for all outbreaks, except for Ob-4 due to the absence of shared well-covered genotype reference sequences.
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