Abstract
BackgroundParechoviruses (PeV-As), which constitute a new genus within the family Picornaviridae, have been associated with numerous localized outbreaks of serious diseases, such as coryza, pneumonia, maculopapular exanthem, and conjunctivitis. However, to the best of our knowledge, only a few laboratories worldwide conduct tests for the identification of this group of viruses. Therefore, in this study, we aimed to develop and validate a real-time RT-PCR assay for the identification of PeV-As.MethodsTo design and validate a real-time PCR primer–probe targeting the 5′-UTR region of PeV-As, the 5′-UTR sequences of PeV-As available in GenBank were aligned using the MUSCLE algorithm in MEGA v7.0. Thereafter, the highly conserved 5′-UTR region was selected, and its primer–probe sequence was designed using Primer Premier v5.0. This primer–probe sequence was then evaluated for specificity, sensitivity, and repeatability, and for its validation, it was tested using fecal samples from 728 healthy children living in Beijing (China).ResultsThe PeV-A real-time RT-PCR assay detected only the RNA-positive standards of PeV-A genotypes (1–8, 14, 17, and 18), whereas 72 serotypes of non-PeV-A EV viruses were undetected. In addition, the VP1 region of these 11 PeV-A genotypes that tested positive were amplified using the primers designed in this study. Typing results indicated that eight, one, and two strains of the 11 were PeV-A1, PeV-A4, and PeV-A6, respectively. We also determined and presented the genetic characterization and phylogenetic analyses results corresponding to these 11 VP1 region sequences. Furthermore, real-time RT-PCR assay showed good sensitivity with LOD of 102 copies/μL. Positive results in eight parallel experiments at each concentration gradient from 107 copies/μL to 102 copies/μL, indicating good repeatability.ConclusionOur findings suggested that the real-time RT-PCR assay developed in this study can be applied for routine PeV-A identification. We detected PeV-A1, 4 and 6 genotypes in the 728 faecal samples using this method. Additionally, we believe that our results will serve as a foundation for further studies on PeV-As and facilitate the expansion of the gene sequence information available in GenBank.
Highlights
Parechoviruses (PeV-As), which constitute a new genus within the family Picornaviridae, have been associated with numerous localized outbreaks of serious diseases, such as coryza, pneumonia, maculopapular exanthem, and conjunctivitis
Real‐time RT‐PCR Specificity testing In this study, performing the real-time reverse transcription polymerase chain reaction (RT-PCR) assay for the identification of PeV-As led to the detection of only the RNA-positive standards of PeV-A genotypes (1–8, 14, 17, and 18), while 72 serotypes of non-PeV-A EV viruses were undetected (Fig. 1)
Sensitivity testing To determine the sensitivity of the PeV-A real-time RTPCR assay, serial dilution of RNA transcripts derived from the clones of the 5′-UTRregion of the 11 PeV-A genotypes (1–8, 14, 17, and 18) was performed, and the corresponding Threshold cycle (Ct) values were recorded (Fig. 2)
Summary
Parechoviruses (PeV-As), which constitute a new genus within the family Picornaviridae, have been associated with numerous localized outbreaks of serious diseases, such as coryza, pneumonia, maculopapular exanthem, and conjunctivitis. In this study, we aimed to develop and validate a real-time RT-PCR assay for the identification of PeV-As. Human parechoviruses (PeV-As), which are RNA viruses that are closely related to enteroviruses of the family Picornaviridae, were first isolated several decades ago [1]. It is known that maculopapular exanthema, which is a common and distinctive rash involving the extremities, with palmar and plantar erythema is associated with PeV-A3 infection (Table 1). To effectively treat such PeV-A-associated diseases, the development of a sensitive and reproducible PeV-A identification method with good specificity is necessary
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