Improved genotyping vaccine and wild-type poliovirus strains by restriction fragment length polymorphism analysis: Clinical diagnostic implications

The combination of preventive vaccination and diagnostic typing of viral is olates from patients with clinical poliomyelitis constitutes our main prote ctive shield against polioviruses. The restriction fragment length polymorp hism (RFLP) adaptation of the reverse transcriptase (RT)-PCR methodology ha s advanced diagnostic genotyping of polioviruses, although further improvem ents are definitely needed. We report here on an improved RFLP procedure fo r the genotyping of polioviruses. A highly conserved segment within the 5' noncoding region of polioviruses was selected for RT-PCR amplification by t he UC53-UG(52) primer pair with the hope that it would be most resistant to the inescapable genetic alteration-drift experienced by the other segments of the viral genome. Complete inter- and intratypic genotyping of poliovir uses by the present RFLP method was accomplished with a minimum set of four restriction endonucleases (HaeIII, DdeI, NcoI, and AvaI). To compensate fo r potential genetic drift within the recognition sites of HaeIII, DdeI, or NcoI in atypical clinical samples, the RFLP patterns generated with HpaII a nd StyI as replacements were analyzed. The specificity of the method was al so successfully assessed by RFLP analysis of 55 reference nonpoliovirus ent erovirus controls. The concerted implementation of these conditional protoc ols for diagnostic inter- and intratypic genotyping of polioviruses was eva luated with 21 clinical samples with absolute success.