Fabio Tummolo

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. J. Med. Virol. 84:643–650, 2012.

Evidence for Recombination between Pandemic GII.4 Norovirus Strains New Orleans 2009 and Sydney 2012

ABSTRACT During 2012, a novel pandemic GII.4 norovirus variant, Sydney 2012, emerged worldwide. A signature of the variant was a GII.Pe ORF1, in association with GII.4 Apeldoorn 2008-like ORF2-ORF3 genes. We report the detection of recombinant GII.4 Sydney 2012 strains, possessing the ORF1 gene of the former pandemic variant New Orleans 2009.

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.

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.

Norovirus GII.4/Sydney/2012 in Italy: winter 2012-2013

To the Editor: Noroviruses (NoVs) are the major cause of acute gastroenteritis in children and adults; they are responsible for sporadic cases and outbreaks of gastroenteritis in various epidemiologic settings. NoVs can be classified genetically into at least 5 genogroups, GI to GV (1). Although >30 genotypes within genogroups GI, GII, and GIV can infect humans (2), a single genotype, GII.4, has been associated with most NoV-related outbreaks and sporadic cases of gastroenteritis worldwide (3). GII.4 NoV strains continuously undergo genetic/antigenic diversification and periodically generate novel strains through accumulation of punctate mutations or recombination. New GII.4 variants emerge every 2–3 years (4). Increased incidence of NoV-related illness and/or outbreaks in various countries in late 2012 has been related to the emergence of a novel GII.4 variant, Sydney 2012. This variant was first identified in March 2012 in Australia (5). The Italian Study Group for Enteric Viruses (ISGEV; http://isgev.net) monitors the epidemiology of enteric viruses in children through hospital-based surveillance (6–8). NoVs are monitored and characterized by multitarget analysis in the diagnostic regions A (open reading frame 1, polymerase) and C (open reading frame 2, capsid) of the NoV genome (9) and interrogation of the Norovirus Typing Tool database (www.rivm.nl/mpf/norovirus/typingtool). During November 2011–March 2012, the prevalence of sporadic NoV infections detected (in samples from newborns, infants, and children up to 5 years of age) by real-time reverse transcription PCR was 22.2% (121/545). A subset (≈50%) of the NoV-positive samples representative of the whole winter period was selected for sequence analysis, and 48 were successfully characterized in region A and region C. Among these 48 NoV strains, 20 (41.7%) were characterized as the variant GII.4 New Orleans 2009, a smaller number, 6 (12.5%), displayed a New Orleans 2009 polymerase (pol) but 2 distinct GII.4 capsid sequences, which were not typeable in the Norovirus Typing Tool database, and only 2 (4.2%) GII.4 strains of the variant Den Haag 2006b were detected. Moreover, 4 sporadic cases in November 2011 and January 2012 and a small outbreak in February 2012 were related to a GII.Pe_GII.4 recombinant strain. After the set of sequences of GII.4 variants from the Norovirus Typing Tool database was updated (access to the updated database: April 11, 2013), 5 (10.4%) GII.Pe_GII.4 recombinant strains were characterized as variant Sydney 2012. From April through October 2012, a total of 56 (7.6%) NoV-positive samples were detected from 737 analyzed samples, of which 34 (60.7%) NoV-positive samples could be sequenced. Of these, 41.2% were characterized as GII.3 (mostly with a GII.Pb pol), 26.5% as GII.Pg_GII.1, and 17.6% as GII.4 variants. From spring to fall 2012, the variant New Orleans 2009 became the predominant GII.4 strain, and the variant Sydney 2012 strain apparently disappeared. During November–December 2012 and January 2013, ISGEV detected NoV infection in 90 (28.9%) of 311 children hospitalized for gastroenteritis. This finding is comparable to a prevalence of 25.2% in the same period (November–January) of the 2011–12 winter season. A representative subset of 45 samples was randomly selected for sequencing, and 26 (74.3%) of 35 fully typed strains were characterized as GII.4 Sydney 2012, which suggested that the new NoV variant had become the predominant strain in Italy. Our findings seem to mirror observations of a report from Denmark that documented the onset and circulation at low prevalence of the variant GII.4 Sydney 2012 at the beginning of 2012 with a marked increase in the prevalence only by the end of 2012 (10). Our surveillance detected the emergence of this variant in Italy at the end of 2011 and provided us with one of the earliest strains of the variant GII.4 Sydney 2012. This novel variant has a common ancestor with the NoV GII.4 variants Apeldoorn 2008 and New Orleans 2009 and has several amino acid changes on the main epitope in the capsid P2 domain (10). Sequence analysis of these early strains of the GII.4 variant Sydney 2012 could help clarify the mechanisms driving its global emergence and spread. Continued surveillance for NoV infections through ISGEV and additional data on clinical and epidemiologic features will enable further assessment of the public health implications of the new variant GII.4 Sydney 2012 in Italy.

Genetic Heterogeneity and Recombination in Human Type 2 Astroviruses

ABSTRACT Novel lineages of human astrovirus (HAstV) types 2, 2c, and 2d have been identified. Upon sequencing of the 3′ end of the genome, the type 2c and 2d HAstVs were found to be open reading frame 1b (ORF1b)-ORF2 recombinant, with ORF1b being derived from type 3 and type 1 HAstVs, respectively. An ORF2 interlineage recombinant strain, 2c/2b, was also identified.

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.

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 early strains of the norovirus pandemic variant GII.4 Sydney 2012 identifies mutations in adaptive sites of the capsid protein.

Global surveillance for norovirus identified in 2012 the emergence of a novel pandemic GII.4 variant, termed Sydney 2012. In Italy, the novel pandemic variant was identified as early as November 2011 but became predominant only in the winter season 2012-2013. Upon sequencing and comparison with strains of global origin, the early Sydney 2012 strains were found to differ from those spreading in 2012-2013 in the capsid (ORF2) putative epitopes B, C and D, segregating into a distinct phylogenetic clade. At least three residues (333, 340 and 393, in epitopes B, C and D, respectively) of the VP1 varied among Sydney 2012 strains of different clades. These findings suggest that the spread of the pandemic variant in Italy during the winter season 2012-2013 was due to the introduction of strains distinct from those circulating at low frequency in the former winter season and that similar strains were also circulating elsewhere worldwide.

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.