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.

FIG. 1.

Schematic representation of the rhinovirus genome. The locations and orientations (arrows) of the primers and the two probes used in the real-time RT-PCR are indicated. Initiation and cleavage sites are defined by the vertical bars, their positions, and the proteins coded for by the reference HRV-2 isolate. The gray bar represents the 5′ NCR from which the consensus sequence, the primers, and the probes were defined.

FIG. 2.

Comparison of the sequences of a 180-bp nucleotide region of the 5′ NCRs of 29 different HRV serotypes. The double-headed arrows represent the locations of the two probes.

FIG. 3.

Phylogenetic tree based on the sequence alignment of a 180-bp nucleotide region of the 5′ NCRs of 107 rhinoviruses. HRV 87 was defined as an outgroup for the classification. The strains of rhinovirus isolates serotyped (n = 30) are in red, clinical rhinovirus isolates (n = 48) are in blue, and nonserotyped isolates obtained from GenBank (n = 29) are in black.

FIG. 4.

Sensitivity of HRV-2 plasmid DNA detection. Serial 10× dilutions of plasmid DNA diluted from l0⁻¹ to 10⁻¹² were tested in duplicate and showed a limit of detection corresponding to 1 plasmid copy /5 μl of reaction mixture.