The analysis of PV strains isolated from vaccinees is an excellent way of studying the evolution of enteroviruses in humans. Studies of PVs isolated from patients with VAPP have demonstrated a high frequency of genetic recombination between OPV strains (9
). A high frequency of intertypic vaccine-derived recombinants was also found in this study by analyzing a total of 88 vaccine-derived PV strains isolated from VAPP cases in Romania. Sequencing combined with RFLP analysis for four different genomic fragments in the regions encoding proteins VP1, 2C, and the 3D polymerase and for the 3′ extremity of the genome showed that 76% of the strains implicated in these cases of VAPP (mostly types 2 and 3) had recombinant genomes. Similar results have been obtained for OPV strains isolated from healthy vaccinees (N. Cuervo et al., unpublished results).
Most of these recombinants were V/V recombinants, but five were shown to contain nonvaccine genomic segments and were classified as V/W recombinants. For one, the nonvaccine parent was identified among the known cocirculating wild PVs. For the others, the wild parent has not yet been identified and the possibility that the wild sequences originate from an NPEV cannot be excluded. Wild PV or NEPV sequences were detected in 6% of the VAPP strains studied. We analyzed wild cocirculating PV strains in an attempt to identify the nonvaccine parents of the V/W recombinants. We detected vaccine-derived sequences in 2 of 15 wild strains. Our findings strongly suggest that there is frequent interchange of genetic material between enteroviruses, whether PVs or NPEVs. Vaccination with OPV creates ideal conditions for this exchange by infecting the gut of the child with three different enterovirus genotypes. In some cases there may already be infection, or superinfection, with another enterovirus genotype.
Given the high frequency of recombinant genomes in the OPV strains excreted by healthy vaccinees (22
; N. Cuervo et al., unpublished results), their contacts in the community, and patients with VAPP (9
; this study), it seems highly likely that genetic recombination is involved in the natural evolution of Sabin strains. OPV strains are reputed to grow less efficiently in the human gut than wild epidemic PVs. Due to the quasispecies properties of PV populations, a large number of variants are likely to arise during the multiplication of vaccine strains in the human digestive tract. Two mechanisms may be responsible for this variation: mutations and recombination. Theoretically, recombination is a more powerful mechanism of variation than mutation because it may transfer a number of properties to the original virus in a single event. The presence of variant populations of PV in the human gut provides a wealth of material for the selection of variants that grow more efficiently.
We determined the neurovirulence of V/W recombinants in PVR-Tg mice and found that none retained the attenuated phenotype of the original Sabin 2 PV. However, the degree of neurovirulence acquired was intermediate between attenuation and the full neurovirulence of wild reference strains. This suggests that recombination alone or in combination with reverse mutations at the attenuating sites of Sabin 2 is not sufficient to render the original vaccine virus highly neurovirulent. This moderate increase in neurovirulence is, however, sufficient to allow Sabin viruses to cause poliomyelitis in man, as demonstrated by the identification of such strains as the etiological agent in certain VAPP cases (12
To estimate the length of time for which the V/W recombinant strains had been circulating, we determined the number of mutations (as a percentage of the total number of bases) accumulated in the vaccine-derived segments. A large number of such mutations probably indicates the circulation of vaccine viruses before and/or after the recombination event. Very small numbers of mutations were found to have accumulated in the Sabin 2-specific regions of the genomes of the six V/W recombinants (from 0 to 0.2% mutated nucleotides). This indicates that recombination probably occurred in the gut of the vaccine recipient or in that of a contact before the disease. The number of mutations accumulated in the Sabin 1 genome during multiplication in humans, with or without disease transmission, has been estimated to be close to 1% per year for the VP1-coding region (16
). However, the frequency of variation may depend on genomic segment and strain. Our results for the Sabin sequences suggest that the W/V recombinants circulated or multiplied for several months before or after recombination with wild PVs.
We cannot determine whether the W sequences in the 3D1 region originated from wild PVs or from NPEVs, directly or via a wild PV, until the donor virus has been positively identified. Four of the five Romanian V/W recombinants were isolated during a period (1980 to 1982) of active circulation of wild PV in Romania. In an attempt to determine the origin of the wild sequences in V/W recombinants, we compared them with homologous sequences from a nucleotide sequence database (GenBank). PV rather than NPEV sequences were selected as the closest match, favoring the hypothesis that silent circulating wild PVs rather than NPEVs were the donors of the wild sequences of the V/W recombinants. This led us to try to identify the wild parent among the cocirculating epidemic PV strains. We looked for similarities in the sequence of the 3D1 region between the recombinants and 61 of 148 wild type 1, 2, and 3 poliovirus isolates. The W sequence in the 3D1 region of strain P2-V/598/80 appeared to be very similar to those of two type 1 wild PVs, isolated several months later in Romania (Fig. ). Phylogenetic analysis of the sequences suggested that the two wild PVs and the donor of the sequence present in the V/W recombinant probably have a common ancestor. A similar situation was found in China (20
), where two P1-V/W recombinants were found in which the W donor was identified as a wild PV circulating in a geographically narrow region. The demonstration that, during their natural multiplication, the PV strains could “trap” sequences from cocirculating wild PV may be used for an active detection of silent wild PV transmission by screening for the V/W recombinants.
However, the donors of the other V/W recombinants described here have not yet been identified among the 61 cocirculating wild PVs tested. Surprisingly, one of the V/W recombinant strains (P2-V/057/87) was isolated in the middle of a 9-year period (1983 to 1991) during which no wild PV was isolated in the surrounding region, under conditions of high-quality virological surveillance. Similarly, in Brazil, a V/W recombinant was isolated from a VAPP case 2 years after the last isolation of a wild PV on the American continent (8
). This suggests that a NPEV rather than a wild PV may have been the donor of the W sequence of the recombinant. Although searches of GenBank mostly showed the most similar sequence to be that of a wild PV, this may be due to the small number of sequences from enteroviruses in the database corresponding to the genomic regions studied here. In the case of the W segments of the V/W recombinants, only the positive identification of an NPEV donor of these sequences could completely exonerate wild PV from being the recombination partner. There is no experimental evidence that recombinants between PV and NPEV are viable. However, there are findings suggesting that such recombinants circulate in nature, as the remarkable similarity to PV of the 3′ end of the CA21 or the CA24 reference strains shows (15
Thus, it is clear that OPV-derived PVs can acquire highly modified genomes not only by mutation but also by genetic exchanges with vaccine or wild PV, or even with NPEV. These PV vaccine-derived strains can survive for long periods of time by prolonged excretion and/or by natural transmission and may cause poliomyelitis in man. If PV eradication is to be successful after OPV is discontinued, we must hope that PV vaccine-derived strains will disappear before they can spread in the growing nonimmune population. This should be carefully studied before a general policy of discontinuing OPV immunization is implemented.