Short communicationRecombinant norovirus GII.g/GII.12 gastroenteritis in children
Highlights
► GII.g/GII.12 norovirus has been recently associated with foodborne outbreaks worldwide. ► In 2009–2010 GII.12 strains caused 16% of the NoV outbreaks in the United States. ► During 2009–2010 we identified GII.g/GII.12 from sporadic enteritis in Italian children. ► Stable circulation of GII.g/GII.12 in the infantile population may facilitate their evolution. ► Phylodynamic analysis demonstrated that GII.g/GII.12 has a high rate of evolution.
Introduction
Noroviruses (NoVs) belong to the Caliciviridae family and are considered as the major cause of acute gastroenteritis in both children and adults. NoVs are responsible for sporadic cases and outbreaks of gastroenteritis in various epidemiological settings, including restaurants, schools, day-care centers, hospitals, nursing homes, and cruise ships. NoV genome includes three open-reading frames (ORFs). ORF1 encodes non-structural proteins including the RNA-dependent RNA polymerase (RdRp), while ORF2 and ORF3 encode the major capsid protein VP1 and minor structural protein VP2, respectively. NoVs are classified into five distinct genogroups (GI through GV) on the basis of the full-length VP1 sequence. GI NoVs infect humans, GII NoVs have been detected in humans and pigs, GIII NoVs circulate in bovines, GIV NoVs infect humans and carnivores, while GV NoVs infect only mice (Green, 2007). Some human and canine NoV strains are not classifiable into any NoV genogroups and could represent a potential novel genogroup (Martella et al., 2009). GI and GII NoV strains have been further classified into at least 8 and 19 genotypes, respectively, but a single genotype, GII.4, has been associated with the vast majority of global outbreaks since the mid-1990s (Bok et al., 2009, Zheng et al., 2006). In the winter season 2009–2010 a novel GII.12 norovirus strain emerged and caused 16% of the norovirus outbreaks in the United States (Vega and Vinje, 2011). Marked increase in the number of non-GII.4 outbreaks was confirmed by the CaliciNet surveillance system, with a GII.12 strain being reported in 14% of the outbreaks reported in 12 states (Vega et al., 2011). Sequence analysis of the US epidemic strain demonstrated its recombinant nature (Vega and Vinje, 2011).
Recombination may create novel chimeric strains bearing ORF1 and ORF2 of different parental origin, thus requiring multiple target (ORF1- and ORF2-based) analysis in order to characterize properly the NoV strains (Ambert-Balay et al., 2005). Epidemiological studies have also revealed heterogeneity in the ORF1 of NoV strains, suggesting that capsid-based classification of NoVs should be implemented with a ORF1-based classification system. Attempts to classify the ORF1 of GII NoVs have been made, in order to designate peculiar NoV strains of epidemiological relevance, although this classification/nomenclature has not been based on precise distance criteria applicable to all NoV strains (Bull et al., 2007, NoroNet, 2011). For consistency with the proposed and widely accepted designation of some recombinant GII NoVs, novel GII ORF1 types have been indicated with letters (a–d) (Bull et al., 2007). The same nomenclature has also been adopted and implemented by the European NoV database (NoroNet, 2011).
In this article, we describe the detection in sporadic cases of diarrhea in Italian children of strains showing the same ORF1/ORF2 combination as the novel recombinant GII.12 strain described in the United States. Upon multi-target sequence analysis, the Italian strains were found to have a recombinant nature and to be related to other GII.g/GII.12 NoVs.
Section snippets
Samples
A total of 1374 faecal specimens were collected from hospitalized children from January 2009 to March 2010. Of these specimens, 615 were collected in Palermo, South of Italy, from children less than 5 years old and 759 in Parma, North of Italy, from children less than 14 years old. Faecal samples were stored at −80 °C upon collection and until use.
Real-time PCR detection
Viral RNA was extracted from 140 μl 10% faecal suspensions using the QIAamp Viral RNA kit according to the manufacturer’s instructions (QIAGEN, Hilden,
Results and discussion
Out of 615 faecal specimens tested in Palermo, 69 (11.2%) NoV-positive samples were identified by the real time PCR. Of these 43 (62.3%) could be amplified by the JV primers for sequence analysis and characterization of the RdRp region. In Parma, 147 of 759 specimens (19.4%) were positive for NoV by real time PCR and 66 (44.9%) were characterized by RdRp sequence analysis. Upon analysis of the RdRp sequences with the automated NoV genotype prediction server Norovirus Genotyping Tool Version
Conclusions
GII.g/GII.12 recombinants were first described in 2008 but they now appear to have reached a worldwide distribution. Assuming that a common ancestor generated all the recently circulating GII.g/GII.12 viruses, in our analysis a high polymorphism (defined as hot-spot patterns) and a high rate of evolution was evidenced in partial ORF2 sequences. A similar evolution rate was obtained for GII.4 NoVs using the full-length ORF2 (Bull et al., 2010). Like influenza virus, GII.4 NoVs are able to
Acknowledgments
The present work was supported by the grants ‘Studio dei meccanismi evolutivi dei calicivirus umani’ (Italian Scientific Research Fund PRIN 2008), ‘Caratterizzazione molecolare di norovirus circolanti nella popolazione pediatrica’ (University of Palermo, Italy, Fondi di Ateneo 2007) and “Epidemiologia molecolare e studio dei meccanismi evolutivi di norovirus” (University of Parma, Italy, Fondi di Ateneo 2008).
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