title: Performance of enterovirus genotyping targeting the VP1 and VP2 regions on non-typeable isolates and patient specimens.
abstract: Culture and serotyping of human enteroviruses are time-consuming and labor-intensive. Targeted nucleic acid sequencing has emerged as a powerful alternative to conventional methods. Many published genotyping assays use two-step reverse transcription and polymerase chain reaction (RT-PCR), nested PCR protocols, and/or reflexive testing algorithms. The performance of a one-step RT-PCR protocol, a more clinically practical approach, was evaluated. The VP1 and/or VP2 region of archived enterovirus isolates (n=36, representing 32 serotypes), patient enterovirus isolates not typeable by immunofluorescence antibodies (n=50), and enterovirus from direct patient specimens (48 cerebrospinal fluid, 2 plasma/serum, 1 blood) were amplified and sequenced for genotype identification. The analytical sensitivity of the genotyping assays was 100-fold less than detection by RT-PCR of the 5'-untranslated region. Thirty-four of 36 archived isolates could be genotyped by combining results of VP1 and VP2 target sequencing. Non-typeable isolates included 17 echovirus 18, 6 enterovirus 68, 6 rhinovirus, and 7 which could not be classified further. From clinical specimens, 23 of 51 (45%) could be identified using VP2 typing and the most common types were coxsackievirus B1, echovirus 30, and echovirus 6. Using a one-step RT-PCR without nesting, most enterovirus isolates and a subset of clinical samples with high viral titer could be genotyped.
authors.affiliation: Department of Pathology, University of Utah School of Medicine, Salt Lake City, UT, United States. rosemary.she-bender@hsc.utah.edu
authors.forename: Rosemary C
authors.initials: RC
authors.lastname: She
authors.forename: Weston C
authors.initials: WC
authors.lastname: Hymas
authors.forename: Edward W
authors.initials: EW
authors.lastname: Taggart
authors.forename: Cathy A
authors.initials: CA
authors.lastname: Petti
authors.forename: David R
authors.initials: DR
authors.lastname: Hillyard
mesh_terms: D002555:Cerebrospinal Fluid / Q000821:virology; D004770:Enterovirus / Q000145:classification* / Q000235:genetics / Q000302:isolation & purification*; D004769:Enterovirus Infections / Q000821:virology*; D006801:Humans; D010949:Plasma / Q000821:virology; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity