Analysis of Enterovirus Genotypes Using Single-Strand Conformational Polymorphism

Kim, Jung Hyun; Lee, Jung Hee; Lee, Je Kue; Bang, Won Ki; Hong, Jung Yon; Kim, Eui Chong

Abstract

BACKGROUND: Epidemics of aseptic meningitis due to enteroviruses has occurred annually in the late spring and early summer season. The early detection of such epidemics is important for the prevention of further infection. Enteroviruses consist of 67 serotypes but one or two serotypes may be the causative agents of epidemics in the year and several different serotypes involve sporadic cases. In order to discriminate epidemics from sporadic infection, the serotype should be determined. We evaluated the significance of the single-strand conformational polymorphism (SSCP) of reverse transcription-polymerase chain reaction (RT-PCR) products of 5'-untranslated region (UTR) for the determination of serotypes.
METHODS
The specimens of patients were cultured with RD cell and the RT-PCR was performed in case of the positive cytopathic effect. For the amplification of 153-bp of 5'-UTR, primers (EN1: 5'-CTC CGG CCC CTG AAT GCG GCT AAT-3'; EN2: 5'-ATT GTC ACC ATA AGC AGC CA-3') were used. The RT-PCR products were denatured with 95% formamide at 95degrees C for 5 min and SSCP was performed. 12.5% polyacrylamide gel (49/1 acrylamide/bis) was made by using Mighty SmallTM SE245 Dual Gel Caster (Hoefer Scientific Instruments Inc., USA). Electrophoresis was done at 10 mA for 1.5 h, and silver nitrate stain was performed. The SSCP patterns were compared with serotypes determined by sequence analysis of VP1 region.
RESULTS
Coxsackievirus A9 (two strains), coxsackievirus A16 (10), coxsackievirus B2 (two), coxsackievirus B3 (two), echovirus 3 (two), echovirus 11 (two), echovirus 16 (seven), echovirus 19 (two), echovirus 30 (three), and enterovirus 71 (six) showed the different SSCP patterns according to their serotypes. The same pattern was observed in the same serotype, except echovirus 30 showing two different patterns.
CONCLUSIONS
The SSCP of RT-PCR products of 5'-UTR may be helpful to determine the serotype and discriminate epidemics from sporadic cases. This has the advantage to be able to test the specimens directly without the viral culture. But the serotype should be determined by other method such as neutralization or sequence analysis in case of the first isolate in the epidemic season and the stains showing the new SSCP patterns.