title: A universal primer-independent next-generation sequencing approach for investigations of norovirus outbreaks and novel variants.
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.
authors.affiliation: Virology Surveillance and Research Section, Department of Microbiological diagnostics and Virology, Statens Serum Institut, Copenhagen, Denmark. FON@ssi.dk.
authors.forename: Jannik
authors.initials: J
authors.lastname: Fonager
authors.affiliation: Department of Microbiology and Infection Control, Statens Serum Institut, Copenhagen, Denmark.
authors.forename: Marc
authors.initials: M
authors.lastname: Stegger
authors.affiliation: Virology Surveillance and Research Section, Department of Microbiological diagnostics and Virology, Statens Serum Institut, Copenhagen, Denmark.
authors.forename: Lasse Dam
authors.initials: LD
authors.lastname: Rasmussen
authors.affiliation: Virology Surveillance and Research Section, Department of Microbiological diagnostics and Virology, Statens Serum Institut, Copenhagen, Denmark.
authors.forename: Mille Weismann
authors.initials: MW
authors.lastname: Poulsen
authors.affiliation: Virology Surveillance and Research Section, Department of Microbiological diagnostics and Virology, Statens Serum Institut, Copenhagen, Denmark.
authors.forename: Jesper
authors.initials: J
authors.lastname: Rønn
authors.affiliation: Department of Microbiology and Infection Control, Statens Serum Institut, Copenhagen, Denmark.|Department of Veterinary Disease Biology, University of Copenhagen, Copenhagen, Denmark.
authors.forename: Paal Skytt
authors.identifier: http://orcid.org/0000-0001-5656-0427
authors.initials: PS
authors.lastname: Andersen
authors.affiliation: Virology Surveillance and Research Section, Department of Microbiological diagnostics and Virology, Statens Serum Institut, Copenhagen, Denmark.|Department of Infectious Diseases and Centre for Global health, Clinical Unit, University of Southern Denmark, Odense, Denmark.
authors.forename: Thea Kølsen
authors.initials: TK
authors.lastname: Fischer
mesh_terms: D017250:Caliciviridae Infections / Q000453:epidemiology / Q000635:transmission / Q000821:virology*; D004196:Disease Outbreaks; D060005:Genotyping Techniques / Q000379:methods*; D059014:High-Throughput Nucleotide Sequencing / Q000379:methods*; D006801:Humans; D029322:Norovirus / Q000235:genetics* / Q000302:isolation & purification; D017422:Sequence Analysis, DNA / Q000379:methods*