Polioviruses with natural recombinant genomes isolated from vaccine-associated paralytic poliomyelitis

To determine how oral poliovirus vaccine (Sabin) strains evolve during replication in humans and to confirm the etiology of vaccine-associated paralytic poliomyelitis (VAPP), we examined 70 vaccine-derived strains isolated from VAPP cases. Two distant sequences of the poliovirus genome were targeted for a double restriction fragment length polymorphism assay (RFLP) of reverse-transcribed genomic segments amplified by PCR, an extension of the method that we described previously (Balanant et al., 1991). One (RFLP-1) was a 480-long nucleotide sequence coding for the N-terminal part of the VP1 capsid polypeptide, situated in the 5' third of the viral genome (nucleotides 2401-2880). The other (RFLP-3D1) was a 291-long nucleotide sequence coding for a part of the viral polymerase, situated near the 3' end of the genome (nucleotides 6086-6376). Strain-specific restriction profiles could be generated for different field isolates by using three restriction enzymes in each case: HaeIII, DdeI, and HpaII for RFLP-1 and HaeIII, DdeI and RsaI for RFLP-3D1. With few exceptions, the vaccine-specific RFLP profiles were found to be conserved in both regions during replication of these viruses in humans. Thus, RFLP could be used as a marker so as to identify the origin of viral isolates at both ends of their genome. Whether viral isolates were vaccine-derived was determined by using strain-specific monoclonal antibodies and RFLP-1. Among the 70 isolates, 21 of the 43 type 2 strains and 15 of the 22 type 3 strains had a recombinant genome. None of the 5 type 1 Sabin-derived isolates was found to be recombinant. Both intertypic vaccine/vaccine and vaccine/non-vaccine recombinants were detected. Partial nucleotide sequencing confirmed the RFLP results in all cases that were investigated. In one case, it was possible to predict the recombination junction site from the restriction profiles. This site was more precisely localized by sequencing. The C6203 > U nucleotide substitution, which is suspected to contribute to the reversion toward neurovirulence of the attenuated Sabin 1 strain, was detected in almost all the recombinant genomes containing Sabin 1-specific sequences at the 3' extremity. This mutation was detected by identification of the modified RsaI profile in the RFLP-3D1. The results presented in this paper suggest that recombination, alone or together with mutation, might be one of the mechanisms of the reversion toward neurovirulence of attenuated vaccine strains and of the natural evolution of poliovirus.