Food and Environmental Virology https://doi.org/10.1007/s12560-019-09402-3 ORIGINAL PAPER Characterization of Norovirus and Other Human Enteric Viruses in Sewage and Stool Samples Through Next‑Generation Sequencing Sofa Strubbia1 · My V. T. Phan2 · Julien Schaefer1 · Marion Koopmans2 · Matthew Cotten2,3,4,5 · Françoise S. Le Guyader1 Received: 5 July 2019 / Accepted: 17 August 2019 © The Author(s) 2019 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-specifc 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 · Norovirus · Sewage · Metagenomic · Virome Introduction Metcalf et al. 1995; Gerba et al. 2018). Documenting viral prevalence and diversity in sewage may be a useful method Increasing human population leads to increased demand for for monitoring viruses circulating in the community (Smith agricultural products and water, wastewater re-use will be et al. 2016). However, their detection by PCR approaches necessary, which will pose a risk for virus pollution of the requires previously identifed sequences for primer design environment and subsequent afects viral transmission (Sano and will not detect more distant viruses. et al. 2016). More than 100 species of enteric viruses have Metagenomics refer to the description of all nucleic acids been identifed in human feces and in sewage such as astrovi- sequences present in a sample (Forbes et al. 2017). Most of ruses, caliciviruses, enteroviruses, enteric adenoviruses, and human enteric viruses have small RNA genomes making rotaviruses (Fernandez-Cassi et al. 2018; Hoque et al. 2019; their detection difcult. Indeed, their relative abundance if compared to bacteria, phage, parasites present in the envi- * Françoise S. Le Guyader ronment challenge their detection and identifcation (Cot- [email protected] ten and Koopmans 2016; Nieuwenhuijse and Koopmans, 2017; Adriaenssens et al. 2018). Thus, viral metagenomics 1 Ifremer, Laboratoire de Microbiologie, LSEM-SG2M, BP is generally performed by removing as much host and bac- 21105, 44311 Nantes Cedex 3, France teria as possible followed by nuclease treatment to remove 2 Department of Viroscience, Erasmus MC, University free nucleic acids (Kim et al. 2017; Nieuwenhuijse et al. Medical Center Rotterdam, Rotterdam, The Netherlands 2017). Detection of viruses can be performed after de novo 3 Present Address: London School of Hygiene and Tropical assembly of short-read data into longer sequences (contigs) Medicine, London, UK followed by a variety of computational methods for detecting 4 Present Address: Uganda Virus Research Institute, Entebbe, known and novel viral sequences (Cotten et al. 2014; Cot- Uganda ten et al. 2016; Oakeson et al. 2017). However, each sample 5 Present Address: MRC-Centre for Virus Research, Glasgow, type is unique and methods need to be adapted to account for UK Vol.:(0123456789)1 3 Food and Environmental Virology origin and to address the metagenomic objective. A concrete example was provided by the analysis of samples collected MethodA MethodB Method C 5 stools 1 stool 3 sewages during the Tara oceans expedition (Alberti et al. 2017). The 4 sewages 4 sewages strategy applied allowed generation of data from a variety of organisms, including viruses and plankton from oceanic samples collected worldwide. Their approach showed the Conductivity and acidic conditions importance of using separate processing steps to analyze the diferent compartments of a volume of (sea)water. To identify human enteric viruses in complex samples such as stool or sewage samples, a method able to select these small PEG precipitation particles resistant to acidic conditions, to eliminate bacteria and to decrease as much as possible background genomes such as phage genomes will be useful. Mucine capture The aim of the current study was to evaluate methods for human enteric virus detection using metagenomics with a focus on norovirus. Noroviruses have great genomic diver- Filtration & Omnicleave treatment sity and they are divided into seven genogroups and many genotypes based upon genomic sequence phylogeny (de Graaf et al. 2017). Three of these genogroups (GI, GII and GIV) infect humans, and constitute the principal agent of Nucleicacidextraction acute gastroenteritis worldwide. Importantly for environ- mental research norovirus are excreted at high concentra- Norovirus tions by infected individuals and they are highly persistent RNA enrichment (Atmar et al. 2018). Their concentrations in sewage, that may vary among countries, are usually high and their pres- ence in waters constitutes a major public health issue nowa- Librairies & Miseqsequencing days (Sano et al. 2016; Schaefer et al. 2018). To specifcally enrich our metagenomics libraries in norovirus sequences, Fig. 1 we selected three protocols that take advantage of known Schematic of sample processing and sequencing used in this study. The three main frames on the top contain the method names properties of these viruses. The frst method uses SureSelect (A, B and C), the number and the type of sample treated with each target enrichment (Agilent) with probes matching human protocol. Key steps common to all protocols are represented in gray norovirus sequences. The second method comprises pH vari- rectangle ations, based on norovirus capsids isoelectric point and on their resistance to both high and low pH. The third method is based on norovirus binding to glycans structures that are and the remaining four samples (sample 1887, 1919, 1920, present in human but also pig mucins. 1927) were analyzed using method B and C. Method A: Polyethylene Glycol Precipitation Materials and Methods and Norovirus SureSelect Target Samples Sewage samples were concentrated using a polyethylene glycol precipitation method (PEG) as previously described Five human stool samples (sample 570, 287, 5, 581, 582) (Lewis et al. 1988; Schaefer et al. 2018). Briefy, 40 mL of positive for norovirus collected between January 2008 and sample were mixed with 10 mL of a 50% PEG 6000 solution October 2016 were used as a 10% suspension in water. Four (Sigma-Aldrich, St Quentin France) (Fig. 1). After gentle samples were analyzed following method B and one sample agitation overnight at 4 °C, the mixture was centrifuged for following method B and C (stool sample 570) (Fig. 1). Seven 1.5 h at 13,500 × g and the pellet was suspended in 3 mL of raw sewage samples were collected from a gathering point 0.05 M glycine bufer (pH 9). After nucleic acid extraction upstream of any process between January 2014 and February as described below, norovirus RNA sequence were enriched 2017 from diferent sewage treatment plants located in the using the SureSelect target enrichment specifc array dur- southern part of Brittany (France). Three sewage samples ing the library preparation (details in the library preparation (sample 1777, 1797, 1854) were analyzed using method A paragraph) (Brown et al., 2016). 1 3 Food and Environmental Virology Method B: Acidic Treatment and PEG Precipitation at 37 °C with 5 Units of Turbo DNAse, (Ambion, Ther- moFisher Scientifc, France) (method B and C). To optimize human enteric viruses recovery, viruses were acid adsorbed to organic or inorganic particles present in Norovirus Quantifcation the samples by increasing the conductivity of stool sus- pension or sewage samples to 2000 µS by addition of 5 M A microfuidic-based digital one-step RT-PCR, that allow sodium chloride at pH 3 (Mullendore et al. 2001), and quantifcation without external calibration curves was per- then concentrated using PEG precipitation as described formed using primers and probes targeting the ORF1-2 above (Fig. 1). After centrifugation, the pellet was sus- region (Polo et al. 2016). Positive and negative controls were pended in 3 mL of 0.05 M glycine bufer (pH 9), and fl- included in each series, and quantifcation was calculated tered through 5, 1.2, 0.45 and 0.22 µM flters (Minisart using the Poisson distribution (QuantStudio™ 3D Analysis NML 17594, NML17593, PES16533, PES16532). Then, Suite™ Cloud Software, version 3.0.3; ThermoFisher). The the fltrate was incubated with 2000 Units of OmniCleave fnal result was expressed as RNA copies/µl. Endonuclease (Epicentre, Madison, USA) for one hour at 37 °C to eliminate free nucleic acids, followed by nucleic Library and Sequencing acid extraction. For method A, cDNA were synthesized using Superscripts II and random primers according to the manufacturer protocol Method C: Porcine Gastric Mucin (PGM) Capture (Life Technologies).