BackgroundHuman noroviruses are a highly diverse group of viruses classified into three of the five currently recognised Norovirus genogroups, and contain numerous genotypes or genetic clusters. Noroviruses are the major aetiological agent of endemic gastroenteritis in all age groups, as well as the cause of periodic epidemic gastroenteritis. The noroviruses most commonly associated with outbreaks of gastroenteritis are genogroup II genotype 4 (GII-4) strains. The relationship between genotypes of noroviruses with their phenotypes and antigenic profile remains poorly understood through an inability to culture these viruses and the lack of a suitable animal model.Methodology/Principal FindingsHere we describe a study of the diversity of amino acid sequences of the highly variable P2 region in the major capsid protein, VP1, of the GII-4 human noroviruses strains using sequence analysis and homology modelling techniques.Conclusions/SignificanceOur data identifies two sites in this region, which show significant amino acid substitutions associated with the appearance of variant strains responsible for epidemics with major public health impact. Homology modelling studies revealed the exposed nature of these sites on the capsid surface, providing supportive structural data that these two sites are likely to be associated with putative variant-specific epitopes. Furthermore, the patterns in the evolution of these viruses at these sites suggests that noroviruses follow a neutral network pattern of evolution.
Noroviruses are a hitherto unsuspected cause of prolonged morbidity and mortality in adults after allogeneic HSCT. The use of reverse-transcriptase polymerase chain reaction to detect high viral load levels in feces distinguishes norovirus gastroenteritis from gut GVHD.
This study describes a method used to determine the diversity of NoVs co-circulating in the community that consisted of the analysis of a limited number of strains collected from outbreaks occurring at different times of the NoV season. The diversity of twenty NoV strains collected from outbreaks occurring at the beginning of each NoV season (September) was compared to the diversity found in the middle (December) and at the end of the season (March). The method was validated through the characterisation of greater numbers of strains at times when novel genotypes or variants were detected. A total of 864 strains from outbreaks of gastroenteritis from the 2003/04, 2004/05 and 2005/06 seasons were genotyped, with the majority of outbreaks occurring in the UK. There was a greater diversity of NoV genotypes at the beginning of two of the three seasons, 2003/04 and 2005/06, when compared to strains circulating at the end of the seasons, and GII-4 NoV strains predominated (>90%) at the end of each season. Data from this study also identified the co-circulation and differentiation of three major GII-4 variants (v2, v3, and v4). Detailed analysis of a larger number of strains throughout each season confirmed that variants emerged, became the predominant circulating strain and were ultimately replaced with another variant selected from a pool of variants. By June 2006, GII-4 v4 (Hu/NoV/Rhyl440/2005/UK) emerged as the predominant GII-4 strain, usurping the previous GII-4 v3 strain [Hu/NoV/Hunter284E/040/AU] to become the commonest co-circulating strain, in the UK in 2006.
Tracking the spread of noroviruses during outbreaks of gastroenteritis is hampered by the lack of sequence diversity in those regions of the genome chosen for virus detection and characterization. Sequence analysis of regions of the genes encoding the RNA-dependent RNA polymerase and the S domain of the capsid does not provide sufficient discrimination between genotypically related strains of different outbreaks. However, analysis of sequences derived from the region encoding the P2 domain showed 100% similarity among strains from the same outbreak and <100% similarity among strains of different outbreaks. The prolonged nature of some hospital outbreaks, links between hospitals, and the introduction of multiple strains of a single genotype associated with an outbreak aboard a cruise ship were determined using this method. This provides a powerful tool for tracking outbreak strains and the subsequent analysis and validation of interventions in a background of multiple introductions of virus strains of the same genotype or genetic cluster.
Amplified-fragment length polymorphism (AFLP) analysis is the name given to a genotypic technique in which adapter oligonucleotides are ligated to restriction enzyme fragments and then used as target sites for primers in a PCR amplification process. The amplified fragments are electrophoretically separated to give strain-specific band profiles. We have developed a single-enzyme approach that did not require costly equipment or reagents for the fingerprinting of strains ofHelicobacter pylori. The method was assessed with 46 isolates of H. pylori from 28 patients, and the results were compared with those from other genotypic tests. The AFLP profiles derived from HindIII fragments differentiated strains ofH. pylori from unrelated individuals and confirmed the common origin of strains in some family members. AFLP analysis was also applied to investigate persistent infection following antibiotic therapy. Overall, the modified technique was relatively rapid and technically simple yet gave reproducible and discriminatory results. AFLP analysis samples variation throughout the genome and is a valuable addition to the existing genotypic fingerprinting methods for H. pylori.
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