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.

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.

Case report: detection of rotavirus RNA in the cerebrospinal fluid of a child with rotavirus gastroenteritis and meningism.

Although case reports have described detection of rotavirus (RV) in extraintestinal sites such as the liver, kidney, and central nervous system (CNS) of children with RV gastroenteritis, CNS localization in RV infection seems to be rare. RT-PCR and nucleotide sequencing detected a G1P[8] strain in the stool and cerebrospinal fluid (CSF) samples of a patient with concurrent RV-associated enteritis and CNS signs. Upon sequence analysis, the viruses detected in the CSF was identical to the virus detected in the stools. In the VP7- and VP4-based phylogenetic dendograms the strain clustered within the G1-Ic sub-lineage and the P[8]-III lineage. This study supports the hypothesis that RV infection was able to spread from the intestinal tract to the CNS, and likely played a role in the onset of neurological disease.

Molecular characterization of group C rotaviruses detected in children in Italy.

BACKGROUND: Group C rotavirus (GCRV) infection has been described worldwide in children and adults either as sporadic cases or large outbreaks of gastroenteritis but GCRV epidemiology is still unclear. OBJECTIVE: To acquire molecular information on GCRV infection in children hospitalized with gastroenteritis in Italy. STUDY DESIGN: Stools positive for rotavirus-like-particles by electron microscopy during the 2004-2005 surveillance for rotavirus infection in Parma, Italy, were screened by polyacrylamide gel electrophoresis. GCRV strains detected were characterized by sequence and phylogenetic analysis of the genes encoding the capsid proteins VP4, VP6 and VP7. RESULTS: Two of 856 samples (0.23%; 0.7% of 273 samples containing rotavirus-like particles) contained GCRV. These Italian strains were virtually identical in the 3 genes and were closely related to human strains identified in Asia, rather than to strains of European origin. CONCLUSIONS: These results confirm the circulation of GCRVs in Europe and demonstrate genetic heterogeneity among European GCRVs. Inclusion of GCRVs in the diagnostic algorithms for childhood diarrhoea could be helpful in monitoring temporal changes in the GCRV epidemiology, likely under the influence of the changing demographic dynamics.

Molecular characterization of VP4, VP6 and VP7 genes of a rare G8P[14] rotavirus strain detected in an infant with gastroenteritis in Italy.

In this study, the molecular characterization of a rare G8P[14] group A rotavirus (GARV) strain detected in Northern Italy during the 2004-2005 epidemiological rotavirus season is described. Two hundred and seventy three rotavirus-like particle positive stools out of 856 stools from children (31.9%) hospitalized with gastroenteritis were analyzed using polyacrilamide gel electrophoresis and 271 GARVs were genotyped by VP7 and VP4 specific RT-PCRs. One strain (PR/1300/04) with a long electropherotype (e-type) displayed the G8 specificity and was VP4 un-typeable. The P and the subgroup (SG) specificities were determined by sequencing the VP4 and the VP6 gene, respectively. The PR/1300/04 strain exhibited P[14] and SGI specificities. By sequence and phylogenetic analyses of the VP4, VP6 and VP7 amplicons, the PR/1300/04 VP4 and VP6 genes were demonstrated to be of human rotavirus origin, with the VP4 gene closely related to the human Italian PA169 strain (G6P[14]), while the VP7 gene was of animal origin (bovine). These data suggest that the Italian PR/1300/04 strain could be a reassortant between a PA169-like Italian strain with P[14] specificity, long e-type and SGI, and a G8 animal strain. The increasing number of reports of atypical GARVs in humans suggests that interspecies transmission of genes greatly contributes to the GARV genetic evolution.

HBV genotypes and antiviral-resistant variants in HBV infected subjects in Northern Italy.

HBV genotypes were investigated in sera/plasma from 97 HBV positive subjects. Genotype D was revealed in 80.4% followed by E in 6.2%. Genotypes A, B, and C were also found, as well as for the first time a new combination of HBV D and G genotypes. In a cohort of subjects of this population, the relationship with lamivudine and/or famciclovir-resistant HBV mutants was also investigated. Among 12 untreated subjects, 25% carried HBV drug-resistant strains suggesting that drug-resistant variants naturally exist in untreated Italian HBV chronically infected subjects.

Epidemiological aspects of human rotavirus infection in children hospitalized with acute gastroenteritis in an area of northern Italy.

Human rotavirus (HRV) is recognized as the most common cause of severe gastroenteritis in children under 5 years of age. Due to the lack of recent reports about the surveillance of HRV infection in Italy, in this study we assessed the prevalence rate of HRV infection on 1,340 stool samples belonging to 1,264 pediatric patients hospitalized with acute gastroenteritis in the period January 2000--December 2002. The stool samples were submitted to virological investigations by electron microscopy (EM) and conventional cell culture, as well as from January 2002 by RT-PCR for norovirus detection. Reovirus-like particles observed by EM were identified by electropherotyping. Single HRV infections were detected in 302 cases (23.9%, ranging from 19.1% in 2000 to 30.2% in 2001). Mixed infections were observed in 28 cases in which HRV was found to be associated with adenovirus in 16 cases (1.3%), with picornavirus in 4 (0.3%), and with norovirus in 8 (2.1% of the 388 cases examined in 2002). The 3 major epidemic periods of HRV infections were March--May 2000 (66 cases), December 2000--May 2001 (128 cases) and September 2001--April 2002 (105 cases) with peaks in March, January and March, and January, respectively. In the periods of major incidence, single HRV infection accounted even for 52.5% of the gastroenteritis cases monthly examined. According to age distribution, 68.9% (208 cases) of HRV infected children was under 4 years (69.6%: 230/330 cases, including mixed infections) and 16.9% (51 cases) was in the 5-12-year age-group. The epidemiological aspects of HRV infection, also compared to other enteric virus infections, will contribute to assess the magnitude of the problem of HRV in different settings and to devise strategies for intervention.

Norovirus RNA in the blood of a child with gastroenteritis and convulsions--A case report.

Potential extra-intestinal spread is an important issue in understanding the pathogenesis of NoV disease. A previously healthy 14-month-old boy was admitted to the Pediatric Emergency Department of the University-Hospital of Parma, Italy, for afebrile convulsions in a gastroenteritis episode. Bacterial culture and microscopic examination on cerebrospinal fluid (CSF) yielded negative results as well as PCRs and reverse-transcription PCRs (RT-PCRs) for neurotropic viruses performed either on CSF or plasma. Stools were subjected to electron microscopy and conventional cell culture, yielding negative results. NoV was found in stools and plasma by nested RT-PCR targeting the NoV polymerase gene. The nucleotide sequences obtained from the two specimens showed 100% identity, demonstrating that the strain invading the blood stream was from the intestine, and, in comparison with GenBank sequences, they belonged to NoV genotype GII.4, "2006b" variant. The child had no abnormal electrolyte balance and no fever that could justify seizures, encouraging the hypothesis that NoV could be the cause of the neurologic disorder. These findings further induce to review the current concept of human NoV focused on intestinal infection.

Characterization of inter-genogroup reassortant rotavirus strains detected in hospitalized children in Italy.

Analysis of archival stool collections provides an invaluable source of virus strains and genetic material that may be exploited for molecular, epidemiological, and biological studies. The aim of this study was the molecular characterization of unusual human rotavirus (HRV) strains displaying atypical combinations of electropherotype (e-type) and VP4 and/or VP7 genotypes. Analysis of a panel of archival stools collected in northern Italy revealed continual circulation of P[8]G1 HRVs during 1987-1990 and the onset of P[6] + P[8]G1 strains after 1989. Interestingly, nine G1 strains, associated with either P[8], P[4] + P[8], P[6] + P[8], or untypeable VP4 genes, and two P[4]G1 + G2 strains, displayed short RNA e-type. The genetic constellation of the unusual strains was investigated by analysis of the VP4, VP6, VP7, and NSP4 genes. All the G1 strains with short e-type were subgroup (SG)II or SGI + SGII, and possessed a NSP4 of genogroup B or A + B. Conversely, the P[4]G1 + G2 strains were SGI and possessed a genogroup A NSP4. Sequence analysis of the VP7 and VP4 genes revealed that the unusual P[8]G1 and P[4]G1 + G2 viruses emerged by reassortment of strains circulating locally, rather than by introduction of new strains.

Molecular epidemiology of norovirus infections in sporadic cases of viral gastroenteritis among children in Northern Italy.

Surveillance of norovirus infections in sporadic cases of pediatric gastroenteritis admitted to a main hospital in Northern Italy during a full-year period (2002) showed that noroviruses (10.4%) were the second most common causative viral agent, following rotaviruses (21.1%), and noroviruses (81%) were mostly implicated in mixed infections. The epidemic period of norovirus was September-December, with September and November as months of major prevalence (33.3 and 38.5%, respectively). Six distinct norovirus genotypes were detected (GI.7, GII.1, GII.2, GII.4, GII.7, GII, not assigned named GIIb), and the predominant genotype was GII.4. A "new GII.4 2002 variant" accounted for 82.9% of total strains. Since the severity of norovirus symptoms does not usually require admission to hospital, the burden of norovirus disease in the general children population may be much higher than that suggested by the present hospital-based investigation.

Broadly reactive nested reverse transcription-PCR using an internal RNA standard control for detection of noroviruses in stool samples.

We developed a nested reverse transcription-PCR (nRT-PCR) for the detection of noroviruses in stools, using random primers for RT, the JV12/JV13 primer pair in the first round of nPCR, and a set of nine inner primers for the second, comprising the reverse sequences of primers SR46, SR48, SR50, and SR52, and five novel oligonucleotide sequences (113-1, 113-2, 115-1, 115-2, and 115-3). The specificity of the nRT-PCR was confirmed by testing 61 stools containing enteric viruses other than noroviruses. In comparative assays on either stools or RNA dilutions from two genogroup I and three genogroup II (GII) norovirus-positive samples, nRT-PCR was always at least as sensitive as RT-PCR and Southern hybridization. With some of the samples tested, the increase in sensitivity was 10-fold or higher. For GII viruses, the detectable range of nRT-PCR was estimated to be 8.4 x 10(4) to 2 RNA viral particles. When used on 85 stools from pediatric patients with acute gastroenteritis negative for viruses by electron microscopy and cell culture, the nRT-PCR detected norovirus in 19 samples (22.3%), while it failed to detect one reference RT-PCR-positive sample containing a Desert Shield strain. Sixteen of the 19 nRT-PCR-positive samples gave concordant results with reference RT-PCR and Southern hybridization, and all with sequence analysis. Partial sequencing of the polymerase region revealed that from January to April 2000 all GII strains except two (Rotterdam- and Leeds-like viruses) formed a tight cluster related to Hawaii virus. The nRT-PCR described could prove suitable for large epidemiological studies and for specialized clinical laboratories performing routine molecular testing.