title: Development of an efficient entire-capsid-coding-region amplification method for direct detection of poliovirus from stool extracts.
abstract: Laboratory diagnosis has played a critical role in the Global Polio Eradication Initiative since 1988, by isolating and identifying poliovirus (PV) from stool specimens by using cell culture as a highly sensitive system to detect PV. In the present study, we aimed to develop a molecular method to detect PV directly from stool extracts, with a high efficiency comparable to that of cell culture. We developed a method to efficiently amplify the entire capsid coding region of human enteroviruses (EVs) including PV. cDNAs of the entire capsid coding region (3.9 kb) were obtained from as few as 50 copies of PV genomes. PV was detected from the cDNAs with an improved PV-specific real-time reverse transcription-PCR system and nucleotide sequence analysis of the VP1 coding region. For assay validation, we analyzed 84 stool extracts that were positive for PV in cell culture and detected PV genomes from 100% of the extracts (84/84 samples) with this method in combination with a PV-specific extraction method. PV could be detected in 2/4 stool extract samples that were negative for PV in cell culture. In PV-positive samples, EV species C viruses were also detected with high frequency (27% [23/86 samples]). This method would be useful for direct detection of PV from stool extracts without using cell culture.
authors.affiliation: Department of Virology II, National Institute of Infectious Diseases, Tokyo, Japan minetaro@nih.go.jp.
authors.forename: Minetaro
authors.initials: M
authors.lastname: Arita
authors.affiliation: Division of Viral Diseases, Centers for Disease Control and Prevention, Atlanta, Georgia, USA.
authors.forename: David R
authors.initials: DR
authors.lastname: Kilpatrick
authors.affiliation: Department of Virology II, National Institute of Infectious Diseases, Tokyo, Japan.
authors.forename: Tomofumi
authors.initials: T
authors.lastname: Nakamura
authors.affiliation: Division of Viral Diseases, Centers for Disease Control and Prevention, Atlanta, Georgia, USA.
authors.forename: Cara C
authors.initials: CC
authors.lastname: Burns
authors.affiliation: WHO National Polio Laboratory, University of Maiduguri Teaching Hospital, Maiduguri, Nigeria.
authors.forename: David
authors.initials: D
authors.lastname: Bukbuk
authors.affiliation: WHO National Polio Laboratory, University of Maiduguri Teaching Hospital, Maiduguri, Nigeria.
authors.forename: Soji B
authors.initials: SB
authors.lastname: Oderinde
authors.affiliation: Division of Viral Diseases, Centers for Disease Control and Prevention, Atlanta, Georgia, USA.
authors.forename: M Steven
authors.initials: MS
authors.lastname: Oberste
authors.affiliation: Division of Viral Diseases, Centers for Disease Control and Prevention, Atlanta, Georgia, USA.
authors.forename: Olen M
authors.initials: OM
authors.lastname: Kew
authors.affiliation: Division of Viral Diseases, Centers for Disease Control and Prevention, Atlanta, Georgia, USA.
authors.forename: Mark A
authors.initials: MA
authors.lastname: Pallansch
authors.affiliation: Department of Virology II, National Institute of Infectious Diseases, Tokyo, Japan.
authors.forename: Hiroyuki
authors.initials: H
authors.lastname: Shimizu
mesh_terms: D000818:Animals; D001483:Base Sequence; D036022:Capsid Proteins / Q000235:genetics*; D002460:Cell Line; D005243:Feces / Q000821:virology*; D006801:Humans; D051379:Mice; D008969:Molecular Sequence Data; D016366:Open Reading Frames; D011051:Poliomyelitis / Q000821:virology*; D017955:Poliovirus / Q000145:classification / Q000235:genetics* / Q000302:isolation & purification; D015203:Reproducibility of Results; D016415:Sequence Alignment; D017422:Sequence Analysis, DNA
chemical_list: D036022:Capsid Proteins