Parechovirus typing in clinical specimens by nested or semi-nested PCR coupled with sequencing

BackgroundThe Parechovirus genus (Picornaviridae) contains two known species, Human parechovirus (HPeV) and Ljungan virus (LV). HPeVs cause a wide spectrum of disease, including meningitis, gastroenteritis, encephalitis, respiratory illness, and neonatal sepsis-like disease. LVs are associated with diabetes and myocarditis in bank voles and have been proposed to cause disease in humans. The ability to rapidly and accurately type parechoviruses is critical to understanding their role in human disease.ObjectivesFor parechovirus molecular typing, we sought to develop reverse transcription, nested polymerase chain reaction (RT-PCR) assays to amplify the sequence encoding the VP1 capsid protein from all known members of the Parechovirus genus.Study designThe assays consist of a two-step RT-PCR with primers flanking VP1 (PCR1), followed by semi-nested PCR2A and PCR2B reactions that produce overlapping amplicons, encompassing the complete VP1 gene, as well as a nested PCR2C that amplifies a shorter internal VP1 amplicon.ResultsAll primer sets are 100% sensitive and 100% specific for the 77 parechovirus culture isolates tested. The semi-nested and nested PCR primer sets are 94% sensitive and 100% specific for detection of parechovirus in original specimens. Viral genotype can be deduced from analysis of amplicon sequences. Parechoviruses of the same type share ≥77% complete VP1 nucleotide sequence identity or ≥87% amino acid identity, while those of different types share ≤73% nucleotide identity and ≤81% amino acid identity.ConclusionsThe PCR primers described here amplify VP1 sequences from all known parechoviruses, providing a sensitive, reliable system for molecular typing directly from original clinical specimens.