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

Abstract

The combination of preventive vaccination and diagnostic typing of viral isolates from patients with clinical poliomyelitis constitutes our main protective shield against polioviruses. The restriction fragment length polymorphism (RFLP) adaptation of the reverse transcriptase (RT)-PCR methodology has advanced diagnostic genotyping of polioviruses, although further improvements are definitely needed. We report here on an improved RFLP procedure for the genotyping of polioviruses. A highly conserved segment within the 5' noncoding region of polioviruses was selected for RT-PCR amplification by the UC(53)-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 polioviruses by the present RFLP method was accomplished with a minimum set of four restriction endonucleases (HaeIII, DdeI, NcoI, and AvaI). To compensate for potential genetic drift within the recognition sites of HaeIII, DdeI, or NcoI in atypical clinical samples, the RFLP patterns generated with HpaII and StyI as replacements were analyzed. The specificity of the method was also successfully assessed by RFLP analysis of 55 reference nonpoliovirus enterovirus controls. The concerted implementation of these conditional protocols for diagnostic inter- and intratypic genotyping of polioviruses was evaluated with 21 clinical samples with absolute success.

FIG. 1

RT-PCR–RFLP analysis of two representative reference poliovirus strains (Table 1) with restriction endonucleases HaeIII, DdeI, HpaII, NcoI, StyI, and AvaI. (A) Sabin 1 type strain; (B) Mahoney strain. Lanes M, HaeIII digest of φX174 DNA used to calculate the apparent sizes (in base pairs) of the various fragments indicated in Table 1; lanes 1, uncut RT-PCR product; lanes 2 to 7, restriction fragments produced by digestion with HaeIII, DdeI, HpaII, NcoI, StyI and AvaI, respectively.

FIG. 2

RT-PCR–RFLP analysis of two representative clinical poliovirus samples (Table 2) with restriction endonucleases HaeIII, DdeI, HpaII, NcoI, StyI, and AvaI. (A) Virus from clinical sample with patient code number 6902, genotyped as Sabin type 1; (B) virus from clinical sample with patient code number 1085, genotyped as the Mahoney strain. Lanes M, HaeIII digest of φX174 DNA used to calculate the apparent sizes (in base pairs) of the various fragments indicated in Table 2; lanes 1, uncut RT-PCR product; lanes 2 to 6, restriction fragments produced by digestion with HaeIII, DdeI, HpaII, NcoI and StyI, respectively. In panels A and B, lanes 7 contain the restriction fragments produced by digestion with AvaI.

FIG. 3

Diagnostic enzymatic combinations. The proposed enzyme replacements are shown in boldface and italics.