Real-time reverse transcription PCR detection of norovirus, sapovirus and astrovirus as causative agents of acute viral gastroenteritis
Introduction
Acute gastroenteritis is a worldwide cause of morbidity and mortality and viral pathogens are the most common cause of gastroenteritis in developed countries (Lopman et al., 2003, McIver et al., 2001, Simpson et al., 2003). Known enteric viral pathogens include two genera of caliciviruses (Norovirus and Sapovirus), astrovirus, adenovirus, and rotavirus.
Noroviruses are members of the Caliciviridae family and are now the most commonly reported cause of outbreaks of non-bacterial gastroenteritis worldwide, with high attack rates among both children and adults, especially in semi-closed communities (Billgren et al., 2002, Vipond et al., 2004, Ward et al., 2000). In 2002, 84% of outbreaks of infectious intestinal disease in Ireland were either confirmed or suspected norovirus (NDSC, 2003). Of these outbreaks, 70% occurred in hospitals and other healthcare settings placing an enormous burden on the healthcare system.
Norovirus strains can be segregated into five separate genogroups (GI, GII, GIII, GIV and GV) on the basis of sequence comparison of the RNA polymerase and capsid regions of the genome. Three groups, GI, GII and GIV are known to infect humans. Within genogroups, norovirus strains can be further divided into genetic clusters, or genotypes. Genogroup II/genotype4 (GII/4, Bristol/Lordsdale group) virus strains are the most predominant worldwide and are endemic in hospitals and long-term care facilities (Noel et al., 1999). Similarly, reports across Europe have identified norovirus GII/4 as the predominant viral strains (Boga et al., 2004, Lindell et al., 2005, Vipond et al., 2004, Waters et al., 2006), with reports of epidemic spread of GII/4 norovirus variants (Lopman et al., 2004, Siebenga et al., 2007). Genogroup I (GI) virus strains mostly commonly cause community-based sporadic infections and generally exhibit much larger genetic diversity with GI/3 (Desert Shield Virus) and GI/4 (Chiba) clusters frequently detected.
Sapoviruses are a distinct genus within the Caliciviridae family (Green et al., 2000). While norovirus causes ‘winter vomiting disease’, sapovirus infection is predominantly a diarrhoeal disease occurring chiefly in sporadic cases but also in outbreaks (Chiba et al., 2000). Sapovirus infections occur mostly in children less than five years old although it has been suggested that a subset of sapoviruses may be associated with disease in all age groups (Robinson et al., 2002).
Genetically, sapoviruses are divided into five genogroups (GI, GII, GIII, GIV and GV), with GI, II, IV and V known to infect humans. Similar to norovirus classification, within genogroups sapovirus strains can be further divided into genotypes, with strains belonging to the GI/1 cluster (Sapporo/82) most frequently being reported (Okada et al., 2002).
Human astrovirus (HAstV) infection has been documented in all age groups but predominantly in young children (Espul et al., 2004, Pang and Vesikari, 1999). HAstVs occurs in both sporadic cases and in outbreaks and astroviruses are reportedly the most common viral agent associated with diarrhoea in immuno-suppressed adults (Cubitt et al., 1999, Grohmann et al., 1993).
HastVs are members of the genus Astrovirus and are classified into eight antigenic serotypes, HAstV 1–8 (McIver et al., 2000, Pang and Vesikari, 1999), with serotype 1 predominating in most countries (De Grazia et al., 2004, Guix et al., 2002). Genotypic classification is based on capsid protein precursor gene sequences, and shows good correlation with serotype classification.
Recently, this laboratory has published a method for the rapid detection of group F adenovirus and group A and C rotavirus and explored its application to detection of gastrointestinal viruses in paediatric patients with vomiting and/or diarrhoea (Logan et al., 2006). This paper reports real-time reverse transcription PCR assays for the detection of the other major causes of viral gastroenteritis, namely the caliciviruses, norovirus and sapovirus, and human astrovirus. The increased detection of these viruses in the 140 paediatric stool samples examined using molecular methods as compared to results obtained following examination by electron microscopy (EM) and preliminary genotyping data, highlight the need for the introduction of molecular methods for the routine definitive diagnosis of viral gastroenteritis.
Section snippets
Specimen collection and electron microscopy
A total of 140 stool samples were collected from patients exhibiting symptoms of diarrhoea and/or vomiting received at the Microbiology laboratory, Our Lady's Children's Hospital from February 2004 to April 2005, aliquoted and stored at −80 °C until analysed using molecular methods. Twenty five stool samples received at the laboratory from paediatric patients not exhibiting symptoms of gastroenteritis were similarly aliquoted and stored and used as controls for the purpose of this study.
Stool
Sequence analysis and primer design
Norovirus sequences (GI, II and IV) were examined at the ORF1-2 junction (RNA dependent RNA polymerase/capsid gene junction), the most conserved region of the norovirus genome (Kageyama et al., 2003). Due to the significant genetic diversity even within genogroups, genogroup specific primers and probes were designed to share 100% sequence identity to an alignment of strain sequences representing the appropriate genotypes (13 sequences for GI, 17 sequences for GII, and two sequences for GIV),
Discussion
This work describes the design and development of highly sensitive molecular assays for the detection of norovirus (GI, II and IV), sapovirus (GI, II and IV), and human astrovirus and application of the assays to the routine detection of gastrointestinal viruses in paediatric patients with vomiting and/or diarrhoea. Of the 140 samples examined, one or more enteric viruses were detected in 53 of the stool samples (38%) by real-time RT-PCR. Less that 8% (10/140) of stool specimens were positive
Acknowledgements
Norovirus GI and human astrovirus (types I, III and IV) RNA, kindly provided by Bas van der Veer and Erwin Duizer, National Institute for Public Health and the Environment, Bilthoven, Netherlands, were used to generate plasmid standards for the optimisation of the real-time PCR assays. The Children's Medical & Research Foundation, Our Lady's Children's Hospital is gratefully acknowledged for their financial support for purchase of items of equipment used in this research.
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2021, Journal of Virological MethodsCitation Excerpt :A ‘week peak’ was defined as the week with the highest number of GE stool samples with confirmed viral infection. At the NVRL in Dublin, stool samples were tested against a panel of viruses (RV, AdV F, SaV, AsV and NoV), by polymerase chain reaction (PCR) for AdV and reverse transcription (RT)-PCR for the RV G strains (G 1 - 4, G9 and G12) and P types ([4], [6] and [8]), Rotarix™ RV strain (Rotarix), NoV GI, NoV GII, SaV and AsV (Tiemessen and Nel, 1996; Kageyama et al., 2003; Oka et al., 2006; Freeman et al., 2008; Logan et al., 2007; Gautam et al., 2016). All the samples were extracted using the Roche MagNA Pure 96 kit as per the manufacturer’s protocol.