title: Amplicon sequencing and improved detection of human rhinovirus in respiratory samples.
abstract: Improved knowledge of the genotypic characteristics of human rhinovirus (HRV) is required, as are nucleic detection assays with the capacity to overcome both the similarities between members of the family Picornaviridae and the wide diversity of different HRV serotypes. The goal of the present study was to investigate the variability and the genotypic diversity of clinical strains circulating in the community. Since most reverse transcription (RT)-PCR assays available cannot differentiate HRV from other members of the family Picornaviridae, we also validated an assay specific for HRV detection. The 5' noncoding regions of 87 different HRV serotypes and clinical isolates were sequenced. On the basis of sequence analysis and phylogenetic determination, we first confirmed that all clinical isolates were HRV. We then validated a real-time RT-PCR assay that was able not only to detect all HRV serotypes and all clinical isolates tested but also to accurately discriminate between rhinovirus and other viruses from the family Picornaviridae. This assay was negative with isolates of coxsackievirus (types A and B), echovirus, enterovirus, parechovirus, and poliovirus, as well as nonpicornaviruses. Among a series of bronchoalveolar lavage specimens, 4% (7 of 161) were positive by culture, whereas 13% (21 of 161) were positive by RT-PCR. In the present study we showed that to specifically identify HRV in clinical specimens, diagnostic assays need to overcome both the diversities and the similarities of picornaviruses. By sequencing the 5' noncoding regions of rhinoviruses recovered from clinical specimens, we designed probes that could specifically detect rhinovirus.
authors.affiliation: Central Laboratory of Virology, Division of Infectious Diseases, Department of Internal Medicine, University Hospitals of Geneva, 24 Rue Micheli-du-Crest, CH-1211 Geneva 14, Switzerland.
authors.forename: Christelle
authors.initials: C
authors.lastname: Deffernez
authors.forename: Werner
authors.initials: W
authors.lastname: Wunderli
authors.forename: Yves
authors.initials: Y
authors.lastname: Thomas
authors.forename: Sabine
authors.initials: S
authors.lastname: Yerly
authors.forename: Luc
authors.initials: L
authors.lastname: Perrin
authors.forename: Laurent
authors.initials: L
authors.lastname: Kaiser
mesh_terms: D020121:5' Untranslated Regions / Q000737:chemistry; D001483:Base Sequence; D001992:Bronchoalveolar Lavage Fluid / Q000821:virology*; D006801:Humans; D008969:Molecular Sequence Data; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012229:Rhinovirus / Q000235:genetics / Q000302:isolation & purification*; D012680:Sensitivity and Specificity
chemical_list: D020121:5' Untranslated Regions