Abstract
Human rhinovirus (HRV) is the major aetiology of respiratory tract infections. HRV viral load assays are available but limitations that affect accurate quantification exist. We developed a one-step Taqman assay using oligonucleotides designed based on a comprehensive list of global HRV sequences. The new oligonucleotides targeting the 5′-UTR region showed high PCR efficiency (E = 99.6%, R2 = 0.996), with quantifiable viral load as low as 2 viral copies/μl. Assay evaluation using an External Quality Assessment (EQA) panel yielded a detection rate of 90%. When tested on 315 human enterovirus-positive specimens comprising at least 84 genetically distinct HRV types/serotypes (determined by the VP4/VP2 gene phylogenetic analysis), the assay detected all HRV species and types, as well as other non-polio enteroviruses. A commercial quantification kit, which failed to detect any of the EQA specimens, produced a detection rate of 13.3% (42/315) among the clinical specimens. Using the improved assay, we showed that HRV sheds in the upper respiratory tract for more than a week following acute infection. We also showed that HRV-C had a significantly higher viral load at 2–7 days after the onset of symptoms (p = 0.001). The availability of such assay is important to facilitate disease management, antiviral development, and infection control.
Highlights
Approximately 50% of respiratory tract infections are associated with HRV2, they are often considered self-limiting without causing respiratory distress and possess little medical significance
We developed an improved real-time reverse transcription-PCR (RT-PCR) assay containing newly designed specific oligonucleotides that correspond to a comprehensive list of global Human rhinovirus (HRV) sequences from all HRV species and types
In comparison to other viruses that have been frequently implicated to cause acute respiratory infection such as the influenza viruses[20,21], HRV are often considered innocuous, nearly half of all respiratory tract infections have been associated with HRV2
Summary
Viremia was associated with a significantly higher nasopharyngeal viral load and more severe disease[10]. Limitations that may hinder accurate quantification were evident in many existing assays, such as the lack of sensitivity to detect a broad array of HRV types[11,13] and the potential risk of overestimation of viral load, due to the usage of random hexamer[16,17]. To overcome such limitations, we developed an improved real-time reverse transcription-PCR (RT-PCR) assay containing newly designed specific oligonucleotides that correspond to a comprehensive list of global HRV sequences from all HRV species and types. The newly developed real-time RT-PCR assay can be used as a detection and quantification tool that may be essential in assessing disease prognosis, monitoring viral suppression during antiviral treatment, and designing infection control measures
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