Prevalence and genetic characterization of caliciviruses among children hospitalized for acute gastroenteritis in the United States

https://doi.org/10.1016/j.meegid.2004.06.010Get rights and content

Abstract

Human calicivirus was the first recognized viral agent causing gastroenteritis in humans. Norovirus (NV) and Sapovirus (SV), two genera within the Caliciviridae family, cause epidemic and endemic acute gastroenteritis in children and adults. The role of these viruses as a cause of sporadic acute gastroenteritis in young children requiring hospitalization is not well established. The aim of this study was to assess the prevalence and genetic diversity of caliciviruses among children hospitalized with symptoms of acute gastroenteritis. Stool samples were collected over 2 years from symptomatic children (N = 1840) up to 5 years of age at three pediatric hospitals in the US. Overall, 156 (8.5%) samples were CV-positive, 131 (7.1%) confirmed by sequencing to be NV and 25 (1.4%) confirmed to be SV. Sequences of RT-PCR-amplified polymerase gene segments were analyzed using distance, maximum likelihood and parsimony algorithms. Phylogenetic analysis of 97 NV sequences showed that seven strains were in genogroup I, 86 strains were in genogroup II and four strains were not in genogroup I, II, or III, likely representing three new NV genogroups IV, VI and VII. Genogroup I and genogroup II strains were in 12 new genetic clusters, three in genogroup I and nine in genogroup II. Within genogroups I and II, most (98%) NV strains were in genetic clusters with no known prototype in GenBank. Phylogenetic analysis of 24 SV strains showed that half grouped with the London/92 strain in one genogroup and the remainder in three other proposed genogroups, one novel. In conclusion, NV and SV were frequent causes of hospitalization for acute gastroenteritis in young children and infecting strains were highly diverse, including newly recognized genogroups and genetic clusters within known genogroups.

Introduction

Caliciviruses that commonly infect humans fall into two genera, Norovirus (NV) and Sapovirus (SV). Strains of these genera can be transmitted by the fecal-oral route, fomite exposure, aerosol spread of vomitus and contaminated food or water (Levy et al., 1998, Taylor et al., 1981). NV and SV cause acute gastroenteritis affecting all ages in different community and institutional settings (Fankhauser et al., 1998). Even with the increased rate of detection of these viruses by reverse transcription, polymerase chain reaction (RT-PCR) assays, little is known about the importance of NV and SV as causes of gastroenteritis episodes in young children that require hospitalization. In the US alone, it has been reported that more than 20 million cases, 220,000 hospitalizations and up to 40 deaths from acute gastroenteritis occur annually among children less than 5 years old (Barnes et al., 1998). The fraction of these illnesses caused by CVs is uncertain.

CVs are small (27–38 nm), non-enveloped viruses with a 7.2–8.3 kb poly-adenylated, positive-sense, single-stranded RNA genome possessing three open reading frames (ORFs). ORF 1 encodes a 5.3 kb non-structural polyprotein that contains the 3D RNA-dependent RNA polymerase region, which is relatively well-conserved among CV strains within the same genus (Jiang et al., 1993). ORF 2 encodes the virus capsid, highly diverse among strains in the same genus, and ORF 3 encodes a minor basic virion protein (VP-2) (Green et al., 2002). Like other RNA viruses, CVs exhibit broad genomic sequence diversity among circulating strains, partially due to the poor template fidelity of their RNA polymerases (Green et al., 2002). Widespread occurrence of divergent strains in symptomatic persons suggests their ready infectivity and easy transmissibility. Our inability to grow them in cell culture, and lack of an animal model, creates a major research obstacle to the characterization of attributes of biologic and pathogenic significance. Therefore, sequencing and phylogenetic analysis have emerged as the major tool for studying genetic relationships that exist among CVs. The ability to identify CV genomic lineages permits tracing of pathogen spread and provides information relevant to the development of some therapeutics and potential vaccines (Estes et al., 2000).

Phylogenetic analysis of the RNA polymerase genomic region shows that each of the NV and SV CV genera can be divided into genogroups and sub-genogroup genetic clusters (Ando et al., 2000, Berke et al., 1997, Schuffenecker et al., 2001). Strains of multiple genetic clusters from NV genogroups I and II (called GI and GII) co-circulate in the same geographic region (Fankhauser et al., 1998, Gonin et al., 2000). GII currently is globally predominant among NV strains (Jiang et al., 1995, Koopmans et al., 2000, Noel et al., 1999) and the predominance of a particular genetic cluster within a genogroup can change over time (Lewis et al., 1997, Noel et al., 1999). Most of this knowledge of strain prevalence comes from the study of strains recovered from outbreaks. Such studies likely underestimate the diversity of circulating strains because reports of outbreaks may be biased toward commonly contaminated vehicles of CV spread. Therefore, the aim of this study was to assess over a 2-year period the prevalence and genetic diversity of CVs among children up to 5 years of age hospitalized for acute gastroenteritis at three different sites in the US.

Section snippets

Patients

Patients were enrolled in a longitudinal prospective manner at three pediatric hospitals: Children's Hospital Medical Center in Cincinnati, Ohio; Children's Hospital of The King's Daughters in Norfolk, Virginia; and Children's Hospital Oakland in Oakland, California. Children 15 days to 4 years of age presenting with diarrhea, vomiting, and/or fever of less than 7 days duration were enrolled 5 days a week for two full years at each site from November 1997 to December 1999. Cases were identified

Characteristics of study population and tested stools

A total of 82,937 patients were admitted to the participating hospitals during the study, of which 16,103 age-eligible patients were screened and 2395 with symptoms of acute gastroenteritis were enrolled (Fig. 1). Clinical features of the enrolled population will be reported elsewhere. Of the 2072 stools collected in the study, 1840 from 1840 patients (89% of stool samples) were available for CV testing. Patients with a stool available were significantly younger and had a lower rate of vomiting

Discussion

This is the first large-scale epidemiological study determining the prevalence and genetic diversity of CV strains among sporadic cases of pediatric gastroenteritis in the US. The results from this study also extend the work of Ando et al. (2000) who proposed genogroup and sub-genogroup genetic cluster limits for NV strains based upon sequence comparisons.

The prevalence of human CV illness (8.5%) determined by RT-PCR amplification of the viral polymerase region was comparable to that observed

Acknowledgments

This work was supported by the unrestricted grant from Wyeth Pharmaceuticals and by the US Public Health Service (RO1AI/HD37093 and N01 AI 45252).

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    Present address: Division of Infectious Diseases, Children's Hospital Medical Center, 3333 Burnet Avenue, Cincinnati, OH 45229, USA.

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