ABSTRACT
Noroviruses are, after rotaviruses, the second most common causative agents of acute gastroenteritis in young children. We studied norovirus genotypes in faecal specimens collected from Finnish children followed-up prospectively in rotavirus vaccine trials. Almost 5000 faecal specimens collected from cases of acute gastroenteritis were examined using reverse transcriptase-PCR. A total of 1172 cases (25% of all acute gastroenteritis) were associated with noroviruses. Of these, 96% were genogroup GII. GII.4 was the most common genotype (46%) throughout the study period but the proportion of this genotype varied in different norovirus epidemic seasons. Additional norovirus genotypes detected were: GII.7 (15%), GII.3 (14%), GII.1 (9%), GII.b (7%), GII.2 (3%), and GI.3 (2%). GII.4 dominated during the following years: 1998-1999 (75%), 2002-2003 (88%) and 2006-2007 (98%) while recombinant genotype GII.b was dominant between 2003 and 2004 (83%). In conclusion, genotypes GII.4 and GIIb have emerged as predominant norovirus genotypes in endemic gastroenteritis affecting young infants and children in Finland.
Subject(s)
Caliciviridae Infections/virology , Feces/virology , Gastroenteritis/virology , Norovirus/genetics , Caliciviridae Infections/epidemiology , Capsid , Capsid Proteins/genetics , Child, Preschool , Disease Outbreaks , Finland/epidemiology , Gastroenteritis/epidemiology , Genotype , Humans , Incidence , Infant , Norovirus/classification , Phylogeny , Polymerase Chain Reaction , RNA, Viral/genetics , RNA-Dependent RNA Polymerase/genetics , Reverse Transcriptase Polymerase Chain Reaction , Seasons , Sequence Analysis, RNAABSTRACT
The ability to conduct advanced functional genomic studies of the thousands of sequenced bacteria has been hampered by the lack of available tools for making high-throughput chromosomal manipulations in a systematic manner that can be applied across diverse species. In this work, we highlight the use of synthetic biological tools to assemble custom suicide vectors with reusable and interchangeable DNA "parts" to facilitate chromosomal modification at designated loci. These constructs enable an array of downstream applications, including gene replacement and the creation of gene fusions with affinity purification or localization tags. We employed this approach to engineer chromosomal modifications in a bacterium that has previously proven difficult to manipulate genetically, Desulfovibrio vulgaris Hildenborough, to generate a library of over 700 strains. Furthermore, we demonstrate how these modifications can be used for examining metabolic pathways, protein-protein interactions, and protein localization. The ubiquity of suicide constructs in gene replacement throughout biology suggests that this approach can be applied to engineer a broad range of species for a diverse array of systems biological applications and is amenable to high-throughput implementation.
Subject(s)
DNA, Bacterial/genetics , Desulfovibrio vulgaris/genetics , Genetics, Microbial/methods , Genome, Bacterial , Genomics/methods , High-Throughput Screening Assays/methods , Artificial Gene Fusion , Gene Deletion , Genetic Vectors , Mutagenesis, Insertional/methods , Recombination, GeneticABSTRACT
The standard diagnosis of rotavirus gastroenteritis is based on the demonstration of rotavirus antigen in stools using an enzyme immunoassay (EIA). In this study, a one-step quantitative RT-PCR (Q-PCR) was used for sensitive detection of rotavirus in diarrheal stools. The primers and TaqMan probe for the Q-PCR were selected from a highly conserved region of the non-structural protein 3 (NSP3) of rotavirus. After validation, the test was applied to study rotavirus EIA positive (N=25) and EIA negative (N=143) stool specimens from cases of acute gastroenteritis of all degrees of severity in a prospective follow-up cohort of infants from 2 months to 2 years of age. Q-PCR detected all 25 EIA positive rotavirus antigens and seven additional cases that were rotavirus EIA negative, i.e. 28% more rotavirus positive cases than identified by EIA. It is concluded that Q-PCR using primers targeted at NSP3 is a rapid and sensitive method for diagnosing acute rotavirus gastroenteritis.
