If you’ve never had norovirus, consider yourself one of the few and fortunate. Norovirus is the most common cause of acute gastroenteritis among adults and children in the United States with symptoms including fever, vomiting and diarrhea. It causes 19–21 million illnesses and contributes to 56,000–71,000 hospitalizations and 570–800 deaths yearly (1). The virus is highly contagious and commonly spread through contaminated food or water. At the end of 2015, norovirus made headlines because a Chipotle restaurant in Boston had to close its doors for several weeks after 136 patrons became sick with the virus.
Norovirus is part of the family of Caliciviridae. The viral genome is organized into 3 open reading frames that encode several structural and nonstructural proteins. Noroviruses are classified into 5 genogroups, of which only GI, GII and GIV infect humans. Each genogroup is further divided into multiple genotypes. GII.4 is currently the most prevalent genotype, responsible for approximately 60% of norovirus outbreaks globally (2). Norovirus is non-enveloped and contains a positive-sense single-strand RNA genome. Recombination is common in norovirus and is a significant driving force in viral evolution. Specifically, genotype GII.4 is extremely recombinant and new strains of this genotype replace old strains approximately every 2–3 years (3). Investigating new strains is needed to figure out the virus’ evolution and global epidemiology.
In the past, studies on norovirus were limited by the scarcity of sensitive detection tools. However, a number of advances, such as next-generation sequencing (NGS), have significantly improved norovirus detection in infected patients and increased the value of epidemiological studies. In contrast to Sanger sequencing, which is often used to type norovirus infection, NGS provides information about the total viral population within an infected individual. Additionally, the direction of transmission may be identified by comparing the viral alleles in patients linked by infection. Integrating deep sequencing techniques into routine virus diagnostics on clinical samples expands the range of viruses that can be detected and enables characterization of the detected viruses.
Most reports of using NGS in norovirus detection and characterization have focused on the most prevalent genotypes, mostly GII.4. A new proof of principle study by Bavelaar et al. shows that NGS is equally applicable in the detection and characterization of other norovirus genotypes (4). The authors performed direct multiplexed whole genome sequencing on fecal samples from patients with gastroenteritis. The study included eight norovirus positive fecal samples (samples 1–8) that had been previously characterized using the Noronet typing tool (5) and fecal samples from two patients known to have gastroenteritis caused by norovirus but with unknown genotypes (samples 9 and 10). Sufficient amounts of RNA were isolated from all samples to perform whole transcriptome sequencing for the detection of RNA viruses using the REPLI-g WTA Single Cell Kit. Complete genomic norovirus sequences were obtained from all samples. Norovirus genotypes could be determined from all clinical samples by phylogenetic analysis performed with the CLC Genomics Workbench. For samples 1–8, genotyping results were the same as the results based on norovirus sequence analysis using the Noronet typing tool. Norovirus detected in samples 9 and 10 was characterized as norovirus Gll.4. The viruses appeared to be identical and epidemiological info revealed that the samples were from patients that were located in close proximity at the same period of time, indicating that patient to patient transmission was likely.
NGS is able to provide information on the microbial make-up of clinical specimens in a single test. In one of the samples in this study, both norovirus and coxsackievirus B1 were detected. Detecting viruses not typically tested for in gastroenteritis can have great clinical utility in cases where the cause of gastroenteritis would otherwise be unknown. Although the sample set was small in this study, the protocol used by the authors to detect and characterize different types of norovirus from clinical specimens was proven reliable, and the results support the utility of NGS in routine diagnostics.
Interested in learning more about whole genome sequencing powered by REPLI-g? Learn more
- 1. http://www.cdc.gov/norovirus/about/overview.html
- 2. Siebenga, J.J.,et al. (2009) Norovirus illness is a global problem: emergence and spread of norovirus GII.4 variants, 2001–2007. J. Infect. Dis. 200, 802.
- 3. Bull, A. (2010) Rapid evolution of pandemic noroviruses of the GII. 4 lineage. PLoS Pathog. 6, 1.
- 4. Bavelaar, H.H. (2015) Whole genome sequencing of fecal samples as a tool for the diagnosis and genetic characterization of norovirus. J. Clin. Virol. 72, 122.
- 5. Kroneman, H. (2011) An automated genotyping tool for enteroviruses andnoroviruses. J. Clin. Virol. 51, 121.
The applications presented here are for research use only. Not for use in diagnostic procedures.