Sentinel hospital-based surveillance for norovirus infection in children with gastroenteritis between 2015 and 2016 in Italy.

Noroviruses are one of the leading causes of gastro-enteric diseases worldwide in all age groups. Novel epidemic noroviruses with GII.P16 polymerase and GII.2 or GII.4 capsid type have emerged worldwide in late 2015 and in 2016. We performed a molecular epidemiological study of the noroviruses circulating in Italy to investigate the emergence of new norovirus strains. Sentinel hospital-based surveillance, in three different Italian regions, revealed increased prevalence of norovirus infection in children (<15 years) in 2016 (14.4% versus 9.8% in 2015) and the emergence of GII.P16 strains in late 2016, which accounted for 23.0% of norovirus infections. The majority of the strains with a GII.P16 polymerase showed a GII.2 capsid genotype (79.5%). Also, a marked circulation of strains with a GII.17 capsid (14.0%) was observed, chiefly in early 2016. The emergence and global spread of non-GII.4 noroviruses pose challenges for the development of vaccine strategies.

Emergence of novel recombinant GII.P16_GII.2 and GII. P16_GII.4 Sydney 2012 norovirus strains in Italy, 2016/2017.

In the winter season 2014/15, the GII.P17_GII.17 norovirus strain Kawasaki 2014 emerged in Italy, cocirculating with pandemic GII.4 strains. In March 2016, molecular investigation identified novel GII.P16 recombinant noroviruses in children with gastroenteritis in Italy. In 43.10% of the genotyped noroviruses GII.P16 strains were identified: 12 were characterized as GII.2 and 13 as GII.4 Sydney 2012 capsid genotypes. The GII.P16 genotype became predominant in January- February 2017 along with an increase in norovirus activity. The capsid gene was characterized as GII.2 or GII.4 Sydney 2012 variant. The emergence of two different recombinant GII.P16 viruses, of which one harboring a pandemic GII.4 capsid sequence, suggests the potential for a future pandemic.

Norovirus GII.17 as Major Epidemic Strain in Italy, 2015-16.

In winter 2015-16, norovirus GII.17 Kawasaki 2014 emerged as a cause of sporadic gastroenteritis in children in Italy. Median patient age was higher for those with GII.17 than GII.4 infection (55 vs. 24 months), suggesting limited cross-protection for older children.

A cluster of Enterovirus 71 subgenogroup C2 in a nursery school, Italy, 2014.

During October 2014, enterovirus (EV) RNA was detected in the stools of four children attending the same class in a nursery school, and hospitalized with mild febrile and vomiting disease in Parma, Italy. Upon sequencing, the viruses were characterized as EV71 subgenogroup C2. Phylogenetic analysis of the four EV71 C2 viruses allowed the distinction of a diverging lineage within subgenogroup C2, containing the Italian EV71 C2 strains and viruses detected in France in 2013. The identification of an outbreak of EV71 C2 in Italy extended information on the geographic diffusion and clinical relevance of these viruses in Europe.

Analysis of the full genome of human group C rotaviruses reveals lineage diversification and reassortment.

Group C rotaviruses (RVC) are enteric pathogens of humans and animals. Whole-genome sequences are available only for few RVCs, leaving gaps in our knowledge about their genetic diversity. We determined the full-length genome sequence of two human RVCs (PR2593/2004 and PR713/2012), detected in Italy from hospital-based surveillance for rotavirus infection in 2004 and 2012. In the 11 RNA genomic segments, the two Italian RVCs segregated within separate intra-genotypic lineages showed variation ranging from 1.9 % (VP6) to 15.9 % (VP3) at the nucleotide level. Comprehensive analysis of human RVC sequences available in the databases allowed us to reveal the existence of at least two major genome configurations, defined as type I and type II. Human RVCs of type I were all associated with the M3 VP3 genotype, including the Italian strain PR2593/2004. Conversely, human RVCs of type II were all associated with the M2 VP3 genotype, including the Italian strain PR713/2012. Reassortant RVC strains between these major genome configurations were identified. Although only a few full-genome sequences of human RVCs, mostly of Asian origin, are available, the analysis of human RVC sequences retrieved from the databases indicates that at least two intra-genotypic RVC lineages circulate in European countries. Gathering more sequence data is necessary to develop a standardized genotype and intra-genotypic lineage classification system useful for epidemiological investigations and avoiding confusion in the literature.

Whole genome sequencing reveals genetic heterogeneity of G3P[8] rotaviruses circulating in Italy.

After a sporadic detection in 1990s, G3P[8] rotaviruses emerged as a predominant genotype during recent years in many areas worldwide, including parts of Italy. The present study describes the molecular epidemiology and evolution of G3P[8] rotaviruses detected in Italian children with gastroenteritis during two survey periods (2004-2005 and 2008-2013). Whole genome of selected G3P[8] strains was determined and antigenic differences between these strains and rotavirus vaccine strains were analyzed. Among 819 (271 in 2004-2005 and 548 in 2008-2013) rotaviruses genotyped during the survey periods, the number of G3P[8] rotavirus markedly varied over the years (0/83 in 2004, 30/188 in 2005 and 0/96 in 2008, 6/88 in 2009, 4/97 in 2010, 0/83 in 2011, 9/82 in 2012, 56/102 cases in 2013). The genotypes of the 11 gene segments of 15 selected strains were assigned to G3-P[8]-I1-R1-C1-M1-A1-N1-T1-E1-H1; thus all strains belonged to the Wa genogroup. Phylogenetic analysis of the Italian G3P[8] strains showed a peculiar picture of segregation with a 2012 lineage for VP1-VP3, NSP1, NSP2, NSP4 and NSP5 genes and a 2013 lineage for VP6, NSP1 and NSP3 genes, with a 1.3-20.2% nucleotide difference from the oldest Italian G3P[8] strains. The genetic variability of the Italian G3P[8] observed in comparison with sequences of rotaviruses available in GenBank suggested a process of selection acting on a global scale, rather than the emergence of local strains, as several lineages were already circulating globally. Compared with the vaccine strains, the Italian G3P[8] rotaviruses segregated in different lineages (5-5.3% and 7.2-11.4% nucleotide differences in the VP7 and VP4, respectively) with some mismatches in the putative neutralizing epitopes of VP7 and VP4 antigens. The accumulation of point mutations and amino acid differences between vaccine strains and currently circulating rotaviruses might generate, over the years, vaccine-resistant variants.

Identification of the novel Kawasaki 2014 GII.17 human norovirus strain in Italy, 2015.

Surveillance of noroviruses in Italy identified the novel GII.17 human norovirus strain, Kawasaki 2014, in February 2015. This novel strain emerged as a major cause of gastroenteritis in Asia during 2014/15, replacing the pandemic GII.4 norovirus strain Sydney 2012, but being reported only sporadically elsewhere. This novel strain is undergoing fast diversification and continuous monitoring is important to understand the evolution of noroviruses and to implement the future strategies on norovirus vaccines.

Epidemiological dynamics of norovirus GII.4 variant New Orleans 2009.

Norovirus (NoV) is one of the major causes of diarrhoeal disease with epidemic, outbreak and sporadic patterns in humans of all ages worldwide. NoVs of genotype GII.4 cause nearly 80-90 % of all NoV infections in humans. Periodically, some GII.4 strains become predominant, generating major pandemic variants. Retrospective analysis of the GII.4 NoV strains detected in Italy between 2007 and 2013 indicated that the pandemic variant New Orleans 2009 emerged in Italy in the late 2009, became predominant in 2010-2011 and continued to circulate in a sporadic fashion until April 2013. Upon phylogenetic analysis based on the small diagnostic regions A and C, the late New Orleans 2009 NoVs circulating during 2011-2013 appeared to be genetically different from the early New Orleans 2009 strains that circulated in 2010. For a selection of strains, a 3.2 kb genome portion at the 3' end was sequenced. In the partial ORF1 and in the full-length ORF2 and ORF3, the 2011-2013 New Orleans NoVs comprised at least three distinct genetic subclusters. By comparison with sequences retrieved from the databases, these subclusters were also found to circulate globally, suggesting that the local circulation reflected repeated introductions of different strains, rather than local selection of novel viruses. Phylogenetic subclustering did not correlate with changes in residues located in predicted putative capsid epitopes, although several changes affected the P2 domain in epitopes A, C, D and E.

Genetic heterogeneity and recombination in type-3 human astroviruses.

Human astroviruses (HAstVs) are important enteric pathogens and can be classified genetically and antigenically into eight types. During molecular surveillance for HAstVs in Italy, sequence analysis of the diagnostic region C (about 400 nucleotide in length), located on the capsid (ORF2) gene, identified a novel type-3 strain. Upon sequencing of the full-length ORF2, the type-3 HAstV strain was characterized as a novel ORF2 genetic lineage, designated as 3c. By converse, in the ORF1b the virus was more similar to type-1 HAstVs, rather than to type-3 strains, suggesting a recombination nature, with the crossover site being mapped to the ORF1b/ORF2 junction region. Region C sequences of similar type-3 HAstV identified from European and extra-European countries were retrieved in the databases, suggesting the global distribution of this novel type-3 lineage.

Genetic diversity in three bovine-like human G8P[14] and G10P[14] rotaviruses suggests independent interspecies transmission events.

The group A rotavirus (RVA) P[14] genotype has been detected sporadically in humans and is thought to be acquired through zoonotic transmission. The present study describes the full-length genome analysis of two G8P[14] and one G10P[14] human RVAs detected in Italy. The strains possessed the typical bovine-like I2-R2-C2-M2-A3/A11-N2-T6-E2-H3 genotype constellation. All the segments of the two G8P[14] RVAs were most closely related to bovine(-like) strains but were relatively distant to each other, suggesting two independent interspecies transmission events. Likewise, the G10P[14] RVA gene segments were most similar to bovine(-like) RVAs but distinct from the G8 strains. The history of these strains probably involved the interspecies transmission of these viruses to humans from an as-yet-unidentified animal host, without evidence of reassortment events involving human RVAs. These results reinforce the potential of animal viruses to cross the host-species barrier, causing disease and increased viral genetic diversity in humans.

Analysis of the ORF2 of human astroviruses reveals lineage diversification, recombination and rearrangement and provides the basis for a novel sub-classification system.

Canonical human astroviruses (HAstVs) are important enteric pathogens that can be classified genetically and antigenically into eight types. Sequence analysis of small diagnostic regions at either the 5' or 3' end of ORF2 (capsid precursor) is a good proxy for prediction of HAstV types and for distinction of intratypic genetic lineages (subtypes), although lineage diversification/classification has not been investigated systematically. Upon sequence and phylogenetic analysis of the full-length ORF2 of 86 HAstV strains selected from the databases, a detailed classification of HAstVs into lineages was established. Three main lineages could be defined in HAstV-1, four in HAstV-2, two in HAstV-3, three in HAstV-4, three in HAstV-5 and two in HAstV-6. Intratypic (inter-lineages) ORF2 recombinant strains were identified in type 1 (1b/1d) and type 2 (2c/2b) with distinct crossover points. Other potential intratypic recombinant strains were identified in type 3, type 5 and type 6. In addition, a type-1b strain with a large insertion (~600 bp) of heterologous RNA in the N-terminal region and a type-6 strain with a large RNA rearrangement in the hypervariable region were identified. The classification scheme was integrated in a novel nomenclature system suitable for designation of HAstV strains.

Novel recombinant GII.P16_GII.13 and GII.P16_GII.3 norovirus strains in Italy.

Novel norovirus strains are continuously emerging worldwide. Molecular investigation and phylogenetic analysis identified GII.P16 recombinant noroviruses from the stools of four Italian children with gastroenteritis. The capsid gene was characterized as either GII.13 or GII.3. The GII.P16_GII.13 Italian strains were closely related to German strains involved in a large outbreak in the second half of 2012 and the Italian strains are the first recorded occurrence of GII.P16_GII.13 in Europe.

Evidence of VP7 and VP4 intra-lineage diversification in G4P[8] Italian human rotaviruses.

Intragenotypic heterogeneity of co-circulating rotaviruses is remarkable. Sequence and phylogenetic analyses of the rotavirus VP7 and VP4 genes were performed on selected human G4P[8] strains identified in Parma, Northern Italy, during 2004-2005 and 2008-2012. All the strains clustered into lineages Ic (VP7) and P[8]-III (VP4) in different subclusters with a nucleotide sequence variation up to 4 %. VP7 and VP4 amino acid sequences of the Italian rotaviruses showed multiple changes with the corresponding reference strains as well as with vaccine viruses in the neutralizing epitopes. There is concern that the progressive intra-lineage diversification in the VP7 and VP4 through the accumulation of point mutations and amino acid differences between vaccine strains and currently circulating rotaviruses could generate, over the years, vaccine-resistant variants.

Lineage diversification and recombination in type-4 human astroviruses.

Human astroviruses (HAstVs) are important enteric pathogens and can be classified genetically and antigenically into eight types. During surveillance of HAstVs in Italy, type-4 HAstVs were detected only sporadically and found to cluster into two distinct genetic groups. Upon sequence analysis of the 3' end of the polymerase gene (ORF1b) and of the full-length ORF2, the 2008 type-4 HAstV strains were characterised as a novel ORF2 genetic lineage, designated as 4c. The 2008 type-4 HAstVs also shared the ORF1b gene with similar HAstV-4c strains detected globally, thus displaying a conserved ORF1b/ORF2 asset. By interrogation of the databases, this novel lineage 4c accounted for 60.8% of the type-4 strains identified worldwide and the vast majority of recent type-4 HAstVs. The 2002 type-4 HAstVs displayed a type-4b ORF2, whereas in the ORF1b they resembled type-1 HAstVs. This inconsistency suggests a possible recombinant origin, with the RNA switch taking place upstream the ORF1b/ORF2 junction region. Also, recombination likely played a role in the diversification of the ORF2 of the three type-4 lineages. Multi-target analysis is required for appropriate characterisation and identification of recombinant HAstVs.

Evaluation of the TRCRtest NV-W for norovirus detection in stools by the Transcription-Reverse Transcription Concerted method.

A novel molecular assay, TRCRtest NV-W, based on a transcription-reverse transcription concerted reaction (TRC) for isothermal amplification and real-time detection of norovirus in stools was assessed and compared with an RT-nPCR. Archived stools positive for either different types or variants of norovirus genogroups I and II or other enteric viruses were used to assess the sensitivity and specificity of the novel assay. The TRC assay was 100% specific since it detected all the noroviruses tested and it did not display cross reactivity with other enteric viruses. When screening a collection of 387 stools with the TRC and RT-nPCR assays, the TRC displayed concordance, sensitivity, specificity, positive and negative predictive values of 96.6%, 81%, 99.7%, 98.1%, and 96.3%, respectively, after retesting the negative specimens. Additional PCRs and/or sequencing, used to understand inconsistent results between TRC and RT-nPCR, confirmed all positive results and did not reveal nucleotide variations in the TRC probe and primers binding sites. The TRC assay may be a rapid and ease of use tool for the detection of noroviruses in clinical virology laboratories even in the face of rapidly evolving noroviruses.

Molecular detection and epidemiology of astrovirus, bocavirus, and sapovirus in Italian children admitted to hospital with acute gastroenteritis, 2008-2009.

Although a number of enteric viruses have been identified in children with acute gastroenteritis, the majority of cases of gastroenteritis remain undiagnosed. In order to provide more insights into the epidemiology of enteric viruses that are not included usually in routine diagnostic tests, cases of childhood sporadic gastroenteritis of unknown etiology requiring hospital admission in Parma, Italy, during 2008-2009, were screened for astrovirus (AstV), sapovirus (SaV), and bocavirus (BoV). The stools of 712 children, negative for rotavirus, norovirus, adenovirus, enterovirus, and reovirus, were examined by PCR or RT-PCR for AstV, BoV, and SaV. The prevalence of AstV, BoV, and SaV in the patients examined was 2.1%, 3.2%, 2.4%, respectively, with the viruses being detected mostly in children <3 years of age. AstV strains were characterized by sequencing as types 1, 2, and 4, with a AstV-1 peak occurring in the 2008 fall-winter season. BoV strains were characterized as types 1, 2, and 3, with BoV-3 circulating more frequently in the 2008 autumn and winter season and BoV-2 during March-April 2009. The most common SaVs were GI.2 and GII.1 while GIV and GV SaVs were detected sporadically. Overall, AstV, BoV, and SaV infections accounted for 7.7% of the sporadic cases of acute gastroenteritis with unknown etiology selected for the study. Different virus types and lineages were found to circulate and temporal peaks of virus activity were also demonstrated, suggesting either small clusters of infections or small outbreaks or epidemics in local population.

Recombinant norovirus GII.g/GII.12 gastroenteritis in children.

Recombinant GII.g/GII.12 norovirus (NoV) strains emerged in 2008 in Australia and subsequently have been associated with gastroenteritis outbreaks worldwide. In the winter season 2009-2010 GII.12 strains caused 16% of the NoV outbreaks in the United States. During 2009-2010 we also identified GII.g/GII.12 strains during surveillance of sporadic cases of gastroenteritis in Italian children. Severity scores were calculated for the GII.g/GII.12 NoV infections using the Vesikari scale and in two out of three paediatric cases they exceeded the median value calculated for concomitant GII.4 infections. Upon sequence analysis, the Italian strains were found to be recombinant viruses and displayed different patterns of nucleotide polymorphisms. Phylodynamic analysis with other GII.g/GII.12 recombinants showed a high rate of evolution, comparable to the rates observed for GII.4 viruses. The mechanisms leading to worldwide emergence of GII.12 NoV strains in 2008-2010 are not clear. Monitoring of GII.12 NoV circulation is necessary to understand these mechanisms of evolution.

Clinical and molecular observations of two fatal cases of rotavirus-associated enteritis in children in Italy.

Two fatal cases of infantile rotavirus enteritis occurred in northern Italy in 2005. Both children were severely dehydrated, and death was related to severe cerebral edema. Histological examination demonstrated extensive damage of the intestinal epithelium, villous atrophy or blunting, and macrophage infiltration. The two rotavirus strains were of the G1P[8] type and the long electropherotype. The 2005 G1P[8] rotaviruses differed in the NSP4, VP3, VP4, and VP7 genes from G1P[8] rotaviruses circulating in 2004, suggesting the onset of a new G1P[8] strain in the local population.