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Ten outbreaks of poliomyelitis caused by pathogenic circulating vaccine-derived polioviruses (cVDPVs) have recently been reported in different regions of the world. Two of these outbreaks occurred in Madagascar. Most cVDPVs were recombinants of mutated poliovaccine strains and other unidentified enteroviruses of species C. We previously reported that a type 2 cVDPV isolated during an outbreak in Madagascar was co-circulating with coxsackieviruses A17 (CA17) and that sequences in the 3′ half of the cVDPV and CA17 genomes were related. The goal of this study was to investigate whether these CA17 isolates can act as recombination partners of poliovirus and subsequently to evaluate the major effects of recombination events on the phenotype of the recombinants. We first cloned the infectious cDNA of a Madagascar CA17 isolate. We then generated recombinant constructs combining the genetic material of this CA17 isolate with that of the type 2 vaccine strain and that of the type 2 cVDPV. Our results showed that poliovirus/CA17 recombinants are viable. The recombinant in which the 3′ half of the vaccine strain genome had been replaced by that of the CA17 genome yielded larger plaques and was less temperature sensitive than its parental strains. The virus in which the 3′ portion of the cVDPV genome was replaced by the 3′ half of the CA17 genome was almost as neurovirulent as the cVDPV in transgenic mice expressing the poliovirus cellular receptor gene. The co-circulation in children and genetic recombination of viruses, differing in their pathogenicity for humans and in certain other biological properties such as receptor usage, can lead to the generation of pathogenic recombinants, thus constituting an interesting model of viral evolution and emergence.

Genetic recombination in RNA viruses was discovered many years ago for poliovirus (PV), an enterovirus of the Picornaviridae family, and studied using PV or other picornaviruses as models. Recently, recombination was shown to be a general phenomenon between different types of enteroviruses of the same species. In particular, the interest for this mechanism of genetic plasticity was renewed with the emergence of pathogenic recombinant circulating vaccine-derived polioviruses (cVDPVs), which were implicated in poliomyelitis outbreaks in several regions of the world with insufficient vaccination coverage. Most of these cVDPVs had mosaic genomes constituted of mutated poliovaccine capsid sequences and part or all of the non-structural sequences from other human enteroviruses of species C (HEV-C), in particular coxsackie A viruses. A study in Madagascar showed that recombinant cVDPVs had been co-circulating in a small population of children with many different HEV-C types. This viral ecosystem showed a surprising and extensive biodiversity associated to several types and recombinant genotypes, indicating that intertypic genetic recombination was not only a mechanism of evolution for HEV-C, but an usual mode of genetic plasticity shaping viral diversity. Results suggested that recombination may be, in conjunction with mutations, implicated in the phenotypic diversity of enterovirus strains and in the emergence of new pathogenic strains. Nevertheless, little is known about the rules and mechanisms which govern genetic exchanges between HEV-C types, as well as about the importance of intertypic recombination in generating phenotypic variation. This review summarizes our current knowledge of the mechanisms of evolution of PV, in particular recombination events leading to the emergence of recombinant cVDPVs.

Poliovirus type 1 neurovirulence is difficult to analyze because of the 56 mutations which differentiate the neurovirulent Mahoney strain from the attenuated Sabin strain. We have isolated four neurovirulent mutants which differ from the temperature-sensitive parental Sabin 1 strain by only a few mutations, using selection for temperature resistance: mutant S137C, was isolated at 37.5°C, S138C5 was isolated at 38.5°C, and S139C6 and S139C10 were isolated at 39.5°C. All four mutants had a positive reproductive capacity at supraoptimal temperature (Rct+ phenotype). Mutant S137C1 induced paralysis in two of four cynomolgus monkeys, and the three other mutants induced paralysis in four of four monkeys. The lesion score increased from the S137C, mutant to the S139 mutants. To map the mutations associated with thermoresistance and neurovirulence, we sequenced all regions in which the Sabin 1 genome differs from the Mahoney genome. The S137C, mutant had one mutation in the 5' noncoding region and another in the 3' noncoding region. Mutant S138C. had these mutations plus another mutation in the 3D polymerase gene. The S139 mutants had three additional mutations in the capsid protein region. The mutations were located at positions at which the Sabin 1 and Mahoney genomes differ, except for the mutation in the 5' noncoding region. The noncoding-region mutations apparently confer a low degree of neurovirulence. The 3D polymerase mutation, which distinguishes S138C. and S139 mutants from S137C1, is probably responsible for the high neurovirulence of S138C5 and S139 mutants. The capsid region mutations may contribute to the neurovirulence of the S139 mutants, which was the highest among the mutants.

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We show that poliovirus (PV) infection induces an increase in cytosolic calcium (Ca2+) concentration in neuroblastoma IMR5 cells, at least partly through Ca2+ release from the endoplasmic reticulum lumen via the inositol 1,4,5-triphosphate receptor (IP3R) and ryanodine receptor (RyR) channels. This leads to Ca2+ accumulation in mitochondria through the mitochondrial Ca2+ uniporter and the voltage-dependent anion channel (VDAC). This increase in mitochondrial Ca2+ concentration in PV-infected cells leads to mitochondrial dysfunction and apoptosis.

Two human neuroblastoma cell lines were persistently infected with poliovirus strains of all three serotypes. In persistently infected IMR-32 cells, which were studied in greatest detail, viral antigens were present in most cells, and over a 9-month period virions were found in the medium at high titers. Persistently infected cells were resistant to superinfection by Sabin 1, 2, and 3 poliovirus but sensitive to coxsackievirus B3. The viruses recovered from persistently infected cells were studied for conservation of epitopes, host cell specificity, and temperature resistance phenotype. The antigenic site 1 carried by the major capsid protein VP1 was modified on the persistent viruses of all three serotypes. This was confirmed for one virus by sequencing the corresponding genomic region in which two mutations were detected. The titers of persistent viruses were 1-3 log10 units higher on IMR-32 cells than on nonneuronal HEp-2 cells, while parental viruses had similar titers on both lines. When thermosensitive viruses were used to initiate the infection, the persistent viruses were found to be thermoresistant at 390C. Together the results indicate that the persistent infection correlated with the selection of highly mutated viral strains. Poliovirus-infected neuroblastoma cell lines thus constitute an in vitro model ofchronic viral infections, which are increasingly implicated in human neural diseases.Poliomyelitis paralyses are caused by poliovirus (PV)-induced necroses of motor neurons (1, 2). A late postpolio syndrome has been described: new focal motor neuron deterioration emerges after an average period of 30 years from initial infection (3, 4). One of the hypotheses proposed to account for this syndrome is a persistent viral infection (3, 4). Several members ofthe picornavirus family are effectively able to induce a persistent infection ofcells in vivo and in vitro (5-11). It is assumed that the selection of viral mutants, viral interference, and interferons play a role in this persistence (12,13). For PV, the viral cytolytic action was shown to be limited by the intercellular matrix (14). Cultures in which only a small fraction of cells were susceptible to PV have been described (15)(16)(17). In a more recent study, Kaplan et al. (18) isolated PV-infected HeLa cell lines blocked at different steps of the virus life cycle which underwent periodic crises with cytopathic effects (CPE).In cell lines of neuronal origin, PV-cell interactions have been studied only in one-step growth experiments during the first 8 hr post infection (p.i.) (19). We report here the establishment of a persistent PV infection of human neuroblastoma cells. Most cells harbored viral antigens and cultures produced virions for months at high titers in the absence of detectable CPE. Persistently infected cell lines and the resulting PV mutants are described.MATERIALS AND METHODS Materials. The Leon 37, vFG68 (20), LSc2ab (S1), P712 Ch 2ab (S2), and P3/Leon 12alb (S3) Sabin strains (21) of PV were used. Another enterovirus, coxsacki...