Molecular detection of HuCVs in stool specimens has become a more common diagnostic procedure of acute gastroenteritis in clinical laboratories (3
). The application of PCR-based methods to screen stool specimens has not only shown that the overwhelming majority of outbreaks of acute gastroenteritis are attributable to NLVs but also that HuCVs also cause numerous cases of sporadic gastroenteritis (35
Our results show that human caliciviruses, mainly GGII of the NLVs, are indeed a major cause of both outbreaks and sporadic cases of acute gastroenteritis in infants and young children in eastern Spain. The prevalence rate detected (14.19%) is similar to that reported by other authors, 14% in Dijon, France (4
), 9% in Melbourne, Australia (21
), and 6.5% in the United Kingdom (35
To our knowledge, this is the first Spanish study in which genogroups and genotypes of Norwalk-like viruses have been investigated as etiologic agents of sporadic gastroenteritis cases in children and in gastroenteritis outbreaks. We found that among viral pathogens rotaviruses are the most frequent agents causing sporadic gastroenteritis cases (25.3%), followed by HuCVs (14.19%). In previous studies, we characterized the genotypes of group A rotavirus and the serotypes of astrovirus detected in our geographical area (6
) as well as the presence of astroviruses in wastewater (28
). Our study confirms that NLVs are the main cause of gastroenteritis outbreaks in Spain, as in other EU countries (4
). Interestingly, although it is well known that group A rotaviruses are the primary cause of sporadic cases of gastroenteritis in children, they are not frequently found as the causative agents of gastroenteritis outbreaks.
The predominance of NLV GGII strains during this period of study agrees with previous reports of a higher prevalence of GGII strains than of GGI (4
). A notable difference has also been found in antibody acquisition to Mexico virus (GGII), to which over 70% of children older than 2 years are seropositive, whereas only 12% of these children had been infected with Norwalk virus (GGI) (27
). Molecular epidemiological studies of outbreaks have revealed that GGII strains dominate GGI strains (11
). The reason for this is unknown, although differences in biological properties, such as virulence, routes of transmission, or stability of the virus in the environment, are possible explanations.
The alignments of the RNA polymerase nucleotide sequences of the seven GGI strains isolated in this study with reference strains of the accepted GGI genotypes showed similarities of less than 85%. This, together with the results of the phylogenetic analysis, suggests that some of these isolates might represent new variants of GGI or even new genotypes. This possibility awaits confirmation by capsid gene sequence analysis.
In our study, SLV strains were rarely detected (only four cases [1.29%]) as a cause of severe gastroenteritis in young children, perhaps reflecting the idea that SLVs cause less severe symptoms than NLVs. Similar results were obtained by Kirkwood and Bishop in Melbourne, Australia (21
), who also consider that SLV-associated gastroenteritis may be clinically milder, not requiring hospitalization.
The number of gastroenteritis outbreaks included in this study does not reflect the real figure of outbreaks which occurred during the period of study, because many of them are not reported to the Regional Health Services. Even so, not all outbreaks are tested for a viral etiology and, consequently, calicivirus infections are underdiagnosed.
Strains isolated from particular outbreaks where highly homogenous and no sequence differences among specimens investigated within one outbreak were found. However, in some specimens, a mixture of sequences was detected, both from patients involved in an outbreak and from sporadic cases of infantile diarrhea. This finding was presumably due to mixed infections with more than one single viral strain. We have not detected any remarkable difference in the viral genotypes producing outbreaks or sporadic cases in the community. It has been suggested that there are two distinct epidemiological patterns of NLV strains, one producing endemic infections (i.e., Grimsby virus strains considered endemic in the United Kingdom) and another causing epidemic infections (Norwalk and Mexico viruses) (17
). Our data do not support such hypotheses, although the number of strains sequenced is limited. The same genotypes were found causing both sporadic cases and gastroenteritis outbreaks in 2000 to 2001, and it seems that multiple viral genotypes are circulating at any given time. Continued NLV surveillance across Europe and investigations of the biological differences between strains are needed in order to clarify their molecular epidemiology.
The genetic relatedness of caliciviruses does not always correspond when regions in different open reading frames are sequenced, this may originate by recombinations in the evolution of caliciviruses (29
). The detection of differences in phylogenetic tree topology between different parts of the genome, like the RNA polymerase and the capsid genes, suggests the existence of the recombination process among HuCVs (18
). To identify recombinants more clearly, one must compare the sequence of different regions of the genome against a panel of well-characterized representative sequences in a database, such as the one created by the EU-funded project on “Foodborne viruses in Europe” to search for matching strains.
Capsid gene sequence analysis can be used to verify the viral genotyping based on the sequence of the polymerase gene fragment produced by the diagnostic PCR. There is a correlation between antigenic grouping based on the use of virus-like particles generated using baculovirus expression systems and genomic grouping based on capsid amino acid sequences (12
). Further characterization of the genetic and antigenic relationships will help the classification of human caliciviruses.
Understanding the real prevalence of these viruses, the mechanisms that lead to the emergence of new viral variants and how they spread within populations will improve the prevention of the diarrheal diseases that they cause.