Real-time reverse transcription PCR detection of norovirus, sapovirus and astrovirus as causative agents of acute viral gastroenteritis

Abstract

The design and development of highly sensitive real-time reverse transcription PCR assays for the detection of norovirus genogroups I, II and IV, sapovirus genogroups I, II and IV, and human astrovirus from stool samples is described.

Examination of 140 stool samples from paediatric patients exhibiting symptoms of diarrhoea and/or vomiting resulted in increased detection levels as compared to examination by electron microscopy. Real-time PCR resulted in a 200% increase in the rate of detection of norovirus as compared to electron microscopy. Only genogroup II noroviruses were detected in the stool specimens and when examined using partial-genotyping primers all were identified as clustering with the genogroup II/4 (Bristol/Lordsdale) cluster. Sapovirus was not detected in any of the stool specimens by electron microscopy while 11% (15/140) of specimens were sapovirus positive by real-time RT-PCR, accounting for 36% of calicivirus diarrhoea. Real-time RT-PCR resulted in a tenfold increase in the rate of detection of astrovirus when compared to detection by electron microscopy with both type 1 and type 4 human astroviruses being detected in circulation.

The results highlight the importance of the introduction of molecular methods for the routine screening of stool samples for causative agents of 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.