Stefania Ramirez

Heterogeneity and Temporal Dynamics of Evolution of G1 Human Rotaviruses in a Settled Population

ABSTRACT A rotavirus sample collection from 19 consecutive years was used to investigate the heterogeneity and the dynamics of evolution of G1 rotavirus strains in a geographically defined population. Phylogenetic analysis of the VP7 gene sequences of G1P[8] human rotavirus strains showed the circulation of a heterogeneous population comprising three lineages and seven sublineages. Increases in the circulation of G1 rotaviruses were apparently associated with the introduction of novel G1 strains that exhibited multiple amino acid changes in antigenic regions involved in rotavirus neutralization compared to the strains circulating in the previous years. The emergence and/or introduction of G1 antigenic variants might be responsible for the continuous circulation of G1 rotaviruses in the local population, with the various lineages and sublineages appearing, disappearing, or cocirculating in an alternate fashion under the influence of immune-pressure mechanisms. Sequence analysis of VP4-encoding genes of the G1 strains revealed that the older strains were associated with a unique VP4 lineage, while a novel VP4 lineage emerged after 1995. The introduction of human rotavirus vaccines might alter the forces and balances that drive rotavirus evolution and determine the spread of novel strains that are antigenically different from those included in the vaccine formulations. The continuous emergence of VP7-VP4 gene combinations in human rotavirus strains should be taken into consideration when devising vaccination strategies.

Norovirus and Gastroenteritis in Hospitalized Children, Italy

Noroviruses were detected in 48.4% of 192 children (<3 years of age) hospitalized for gastroenteritis in Palermo, Italy, during 2004; predominant genotypes were GGIIb/Hilversum and GGII.4 Hunter. Of children with viral enteritis, 19.6% had a mixed norovirus-rotavirus infection. The severity of infection was lower for norovirus than for rotavirus but increased in co-infection.

Canine-origin G3P[3] rotavirus strain in child with acute gastroenteritis.

Infection by an animal-like strain of rotavirus (PA260/97) was diagnosed in a child with gastroenteritis in Palermo, Italy, in 1997. Sequence analysis of VP7, VP4, VP6, and NSP4 genes showed resemblance to a G3P[3] canine strain identified in Italy in 1996. Dogs are a potential source of human viral pathogens.

Emerging GII.4 Norovirus Variants Affect Children With Diarrhea in Palermo, Italy in 2006

Although the genetic/antigenic heterogeneity of human noroviruses (NoVs) is impressive, a few genogroup II strains of genotype 4 (GII.4) are dominant worldwide. GII.4 NoVs evolve rapidly and in the last 15 years six epidemic variants have been identified. In 2005–2006, surveillance of sporadic viral gastroenteritis in children in Palermo, Italy, resulted in the detection of NoV strains in 20.9% of the patients admitted to hospital. By restriction fragment length polymorphism (RFLP) and sequence analysis of region A in the RNA‐dependent RNA‐polymerase (RdRp) gene, 59 NoV strains were successfully characterized. Eighty‐one percent of the strains were characterized as GII.4, 14% as GIIb/Hilversum and 5% as GI.1. Phylogenetic analysis of region A and of the ORF1/ORF2 overlapping region of the GII.4 strains recovered in Palermo in the years 2002–2006 revealed the sequential emergence of four variants, GII.4 2002, 2004, 2006a, and 2006b. The variant GII.4 2006a was detected in June and July, 2006, while the variant 2006b first appeared in August, 2006, becoming predominant thereafter. Based on these findings, the dynamics of replacement and circulation of the GII.4 NoV variants in Italy in 2005–2006 appear to have matched the temporal pattern observed in Europe during the same period. J. Med. Virol. 81:139–145, 2009.

Diversity of human rotaviruses detected in Sicily, Italy, over a 5-year period (2001-2005)

SummaryRotavirus infection was detected in 39.9% of 1030 children hospitalized with gastroenteritis in Palermo, Italy, in the period 2001–2005. Rotavirus strains belonging to G1, G4 and G9 types were continually detected, with G1 being the most common type in 2001, 2002 and 2004. A G4 epidemic occurred in 2003, while G9 was predominant in 2005. G2 strains displayed a low prevalence, except in 2003. G3 rotaviruses accounted for 2.7–17% of the gastroenteritis episodes in 2002–2005. The P-type of a subset of 166 strains confirmed the circulation of the usual G/P combinations, but single G1P[6], G9P[9] and G6P[9] strains were also found.

Rare AU-1-Like G3P[9] Human Rotaviruses with a Kun-Like NSP4 Gene Detected in Children with Diarrhea in Italy

ABSTRACT Three G3P[9] rotaviruses, detected in children hospitalized with gastroenteritis in Palermo, Italy, were found to be genetically related to strains of either human or feline origin in the VP7, VP4, and VP6 genes. In contrast, in the NSP4 gene the viruses resembled G2P[4] human strains, suggesting a reassortment between AU-1-like and Kun-like strains.

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.