Characterization of Norovirus and Other Human Enteric Viruses in Sewage and Stool Samples Through Next-Generation Sequencing

Abstract

This study aimed to optimize a method to identify human enteric viruses in sewage and stool samples using random primed next-generation sequencing. We tested three methods, two employed virus enrichment based on the binding properties of the viral capsid using pig-mucin capture or by selecting viral RNA prior to library preparation through a capture using the SureSelect target enrichment. The third method was based on a non-specific biophysical precipitation with polyethylene glycol. Full genomes of a number of common human enteric viruses including norovirus, rotavirus, husavirus, enterovirus and astrovirus were obtained. In stool samples full norovirus genome were detected as well as partial enterovirus genome. A variety of norovirus sequences was detected in sewage samples, with genogroup II being more prevalent. Interestingly, the pig-mucin capture enhanced not only the recovery of norovirus and rotavirus but also recovery of astrovirus, sapovirus and husavirus. Documenting sewage virome using these methods provides information for molecular epidemiology and may be useful in developing strategies to prevent further spread of viruses.

Keywords: Human enteric viruses; Metagenomic; Norovirus; Sewage; Virome.

Fig. 1

Schematic of sample processing and sequencing used in this study. The three main frames on the top contain the method names (A, B and C), the number and the type of sample treated with each protocol. Key steps common to all protocols are represented in gray rectangle

Fig. 2

Heat map of larger viral contig yield. Quality-controlled short-read data were de novo assembled into large contigs and contigs were classified at the protein level by comparison with virus family-specific reference sets (see Methods). The numbers of contigs in each sample with > 60% protein identity and 500 nt minimum contig length were plotted in a heat map. The colorbar to the right indicates number of contigs detected per sample in each virus family

Fig. 3

Scatter plot of reads identified for each of 8 virus families (percentage of total reads). Quality-controlled reads for each of the 18 sample/conditions were mapped to virus family reference sets (see Methods) for Astroviridae, Caliciviridae, Nodaviridae, Leviviridae, Microviridae, Picornaviridae, Picobirnaviridae and Reoviridae. The number of reads identified in each family was plotted as percentage of the total quality-controlled reads for that read set. Data sets were separated into sample types (stool or sewage) and colored according sample treatment (method A: pink, method B: orange and method C: blue)