Valentina Rotolo

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.

Molecular characterization of genotype G6 human rotavirus strains detected in Italy from 1986 to 2009.

Group A human rotavirus (HRV) strains with a bovine-like (G6) major outer capsid protein VP7 were first detected in Palermo, Italy, in the late 1980s, and subsequently worldwide. During a 25-year rotavirus surveillance period, additional HRV G6 strains, associated with either a P[9] or P[14] VP4 genotype, have been detected sporadically, but repeatedly, in Palermo. Whether these G6 HRVs were transmitted to humans directly from an animal reservoir or could have circulated at low prevalence in susceptible individuals is uncertain. Upon sequence analyses of the VP7, VP4, VP6, NSP4 and NSP5 gene segments, all the Italian HRV strains displayed a conserved genotype constellation, G6-P[9]/[14]-I2-E2-H3. Intra-genotypic lineages and/or sub-lineages were observed among the various HRV strains, with some lineage/sublineage combinations being retained over time. Interestingly, two epidemiologically unrelated G6P[9] viruses, collected in the same rotavirus season, were found to have a clonal origin. In conclusion, our results indicate not only diverse origin of animal derived G6 HRVs in Palermo but also suggest human-to-human transmission of certain strains.

Surveillance of human astrovirus circulation in Italy 2002-2005: emergence of lineage 2c strains

By screening faecal samples collected over four consecutive years (2002-2005) from hospitalized children with diarrhoea in Palermo, Italy, astroviruses (HAstVs) were detected in 3.95% of the patients. The predominant type circulating was HAstV-1 but, in 2002, only HAstV-2 and -4 were identified. Interestingly, the HAstVs-2 detected appeared to be consistently different in 5′ end of their open reading frame 2 from the previously described subtypes. These novel type 2 strains were included in a new 2c lineage based on the phylogenetic analysis and the presence of nine peculiar substitutions.

Investigation and control of a Norovirus outbreak of probable waterborne transmission through a municipal groundwater system

During March 2011 an outbreak of gastroenteritis occurred in Santo Stefano di Quisquina, Agrigento, Sicily, Italy. Within two weeks 156 cases were identified among the 4,965 people living in the municipality. An epidemiological investigation was conducted to characterize the outbreak and target the control measures. A case was defined as a person developing diarrhea or vomiting during February 27-March 13, 2011. Stool specimens were collected from 12 cases. Norovirus (NoV) genotype GII.4 variant New Orleans 2009 was identified in stool samples from 11 of 12 cases tested (91.7%). Epidemiological investigations suggested a possible association with municipal drinking water consumption. Water samples from the public water system were tested for NoV and a variety of genotypes were detected during the first 3 months of surveillance, including GII.4 strains belonging to different variants from that involved in the gastroenteritis outbreak. Contamination of the well and springs supplying the public water network was eventually thought to be the source of the NoV contamination.

Innovative and Integrated Strategies: Case Studies

In this chapter, case studies related to biodeterioration, bioaerosol, biocide and biocleaning are reported. The aim is highlighting the role of biology and biotechnology tools for the preventive conservation of organic and inorganic artifacts, understanding how traditional as well as innovative methods can help the conservationists to develop integrated strategies considering works of art/environment/humans as a dynamic system. Particularly, based on the experience acquired during the researches of Laboratory of Biology and Biotechnology for Cultural Heritage (LaBBCH), the authors suggest several approaches to reveal and identify biological systems able to induce biodeterioration of cultural assets, also focusing on bioaerosols in indoor environment to assess the risk for historical-artistic collections. Finally, novel bioactive molecules have been applied to perform biocleaning protocols or to control of microbial colonisation, in accordance to conservative restoration procedures and safety for both the environment and operators.

Variant GII.4 noroviruses in Italian children

Objectives: The analysis of nucleic acids by mass spectrometry (MS) has evolved to a user friendly technology for characterising DNA, and RNA in clinical research and molecular medicine. Recently, the technology has become a versatile tool for microbial identification utilising comparative sequence analysis as shown for 16S based typing of mycobacteria [1]. Here, we present examples for the development of MS specific assays for molecular typing of the M. tuberculosis Complex including spoligotyping and antibiotic resistance identification, which is essential for epidemiological analysis. One technology, the MassARRAY® platform can be used with different assay formats and typing schemes to obtain results from species to strain identification in an automated fashion. Methods: Nucleic acid analysis by MS is based on PCR amplifications using unique primer sets. Traditional spoligotyping detects the presence or absence of 43 different spacer sequences. For MS 43 spacer oligonucleotide probes were designed and the presence of a spacer is detected by base extension (TypePLEXTM). Resistance regions are amplified by PCR with a tagged primer system in multiplex followed by in vitro transcription of both DNA strands. Subsequent endonuclease digests of the RNA transcripts at the bases cytosine and uracil result in four mixtures of RNA cleavage products (iSEQTM). Resistance is identified by correlating acquired spectra with theoretical peak patterns predicted for in silico cleavages of sequences contained in a reference database. Microheterogeneities are identified and deliver new resistance types. Results: Over 200 characterised strains from different reference centres representing the major M. tuberculosis Complex lineages were run over the established spoligotyping and resistance assays. Results were in concordance with traditional spoligotyping and dideoxy sequencing data. Advantages of the MS approach are the homogeneous assay formats without any clean-up steps, semi-automated processing, the time-to result with a throughput of 192 samples in 8 hours for spoligotyping and plexing capabilities for comparative sequence analysis of multiple genomic regions in one reaction. Conclusion: Mass spectrometry specific assay formats for genotyping and comparative sequence analysis generate highly accurate qualitative and quantitative data and provide a toolbox for molecular typing of microbes and viruses.