title: Rapid RT-PCR amplification of full-length poliovirus genomes allows rapid discrimination between wild-type and recombinant vaccine-derived polioviruses.
abstract: Poliomyelitis outbreaks in areas that were free for a long time of wild-type polioviruses have been reported. Characterization at nucleotide level of the causative agents showed that the isolated viruses were recombinant oral polio vaccine (OPV)-derived polioviruses. To allow rapid identification and detailed analysis of such recombinant polioviruses, a robust full-length reverse transcriptase-PCR (RT-PCR) was developed using SuperScript II (RT) and expand (PCR). Without extensive purification, it was possible to amplify and characterize the full-length genomes of all selected vaccine, wild-type, and recombinant vaccine-derived polioviruses within a week. Endonuclease nuclease analysis (SpeI) of the full-length amplicons allowed easy discrimination between recombinant and non-recombinant polioviruses. Furthermore, sequence analysis of cloned full-length amplicons of a recombinant vaccine-derived poliovirus strain showed that the quasi-species nature of a viral stock is preserved during the RT-PCR procedure. This robust and rapid RT-PCR method will allow rapid characterization of (recombinant) poliovirus strains in case of a local poliomyelitis outbreak, and will help to assess the risk of the appearance of such strains after wild-type poliovirus has been eradicated globally.
authors.affiliation: Laboratory of Vaccine-preventable Diseases, National Institute for Public Health and the Environment (RIVM), PO Box 1, 3720 BA Bilthoven, The Netherlands. hein.boot@rivm.nl
authors.forename: Hein J
authors.initials: HJ
authors.lastname: Boot
authors.forename: Rutger M
authors.initials: RM
authors.lastname: Schepp
authors.forename: Femke J H B
authors.initials: FJ
authors.lastname: van Nunen
authors.forename: Tjeerd G
authors.initials: TG
authors.lastname: Kimman
mesh_terms: D005814:Genes, Viral; D016679:Genome, Viral; D006801:Humans; D011051:Poliomyelitis / Q000453:epidemiology / Q000821:virology; D017955:Poliovirus / Q000145:classification / Q000235:genetics* / Q000302:isolation & purification*; D011055:Poliovirus Vaccine, Oral; D012150:Polymorphism, Restriction Fragment Length; D012367:RNA, Viral / Q000032:analysis / Q000235:genetics*; D011995:Recombination, Genetic; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*; D014614:Vaccines, Synthetic / Q000235:genetics / Q000821:virology
chemical_list: D011055:Poliovirus Vaccine, Oral; D012367:RNA, Viral; D014614:Vaccines, Synthetic