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Since the announcement of the WHO program for the global eradication of poliomyelitis and the establishment of epidemiological and virological surveillance, the emergence and circulation of pathogenic vaccine-derived polioviruses (VDPV) presenting >1% nucleotide divergence from the sequence of the original vaccine strain have been demonstrated in certain regions. We developed and used a multiple restriction fragment length polymorphism (RFLP) method to investigate the frequency of these VDPV in a population with a high level of oral poliovirus vaccine coverage in northwestern Russia. Modified RFLP profiles were found to be strongly correlated with the presence of mutations and recombination events in vaccine strains. We found that a substantial proportion of vaccine strains had high percentages of nucleotide substitutions (>0.5%), including a type 3 VDPV with 1.4% nucleotide divergence. These findings indicate that VDPV or pre-VDPV strains are not rare in certain populations with high levels of vaccine coverage. The multiple RFLP method appears to be a simple and rapid tool for monitoring such strains, which could jeopardize the benefits of the eradication program.

The molecular and antigenic properties of a Sabin-like type 2 poliovirus, isolated from the stool samples of a 2-year-old agammaglobulinaemic child who developed paralysis 1 year after receiving the third dose of oral poliovirus vaccine, were analysed. The virus revealed 0.88 % genome variation in the VP1 region compared with the standard reference strain, compatible with replication of the virus in the intestine over approximately 1 year. The typical mutations in the 5'NCR and VP1 associated with reversion to neurovirulence for Sabin type 2 poliovirus were found. Despite this, the virus was characterized by both PCR and ELISA tests as Sabin-like and showed temperature sensitivity and neurovirulence in transgenic mice typical of the Sabin type 2 vaccine strain. Gammaglobulin replacement therapy led rapidly to virus clearance, which, when combined with treatment with the antiviral drug pleconaril, stopped virus excretion; no further virus shedding occurred. This is the first case of poliomyelitis and long-term excretion from an immunodeficient patient to be reported in Italy through the active 'Acute Flaccid Paralysis' surveillance system.

Background Genetic characterisation of polioviruses remains highly important even in countries where wild poliovirus circulation has been interrupted. Sequence data on representative wild strains from all geographical regions is required for surveillance purposes and surveillance for vaccine-related isolates with increased potential for transmissibility in humans should continue. Objective To report the genetic characteristics of wild and vaccine-related polioviruses isolated in Tunisia from 1991 to 2006. Study design Wild isolates were sequenced in the VP1 genomic region and compared to each other. Vaccine-related isolates were assessed for genetic recombination by PCR/RFLP and sequence analysis of the 3D region. Recombinant viruses were assessed for genetic drift in the VP1 region. Results The VP1 sequences of the last wild isolates, all from serotype3, showed 97.7–98.7% nucleotide homology. Nineteen percent of vaccine-related isolates were vaccine/vaccine intertypic recombinants. No recombinant with non-poliovirus enteroviruses was identified. Mutational differences in the VP1 sequences of recombinant viruses ranged from 0.0% to 0.7% indicating a limited replication period. Conclusions This study provides sequence data on wild polioviruses from Tunisia/North Africa and shows that in countries with continuous high vaccine coverage transmission of vaccine-related polioviruses is time-limited.

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 S(1)37C1 was isolated at 37.5 degrees C, S(1)38C5 was isolated at 38.5 degrees C, and S(1)39C6 and S(1)39C10 were isolated at 39.5 degrees C. All four mutants had a positive reproductive capacity at supraoptimal temperature (Rct+ phenotype). Mutant S(1)37C1 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 S(1)37C1 mutant to the S(1)39 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 S(1)37C1 mutant had one mutation in the 5' noncoding region and another in the 3' noncoding region. Mutant S(1)38C5 had these mutations plus another mutation in the 3D polymerase gene. The S(1)39 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 S(1)38C5 and S(1)39 mutants from S(1)37C1, is probably responsible for the high neurovirulence of S(1)38C5 and S(1)39 mutants. The capsid region mutations may contribute to the neurovirulence of the S(1)39 mutants, which was the highest among the mutants.

International audience; The goal of the World Health Organization is to stop routine use of oral poliovirus vaccine shortly after interruption of wild poliovirus transmission. A key component of this goal is to minimize the risk of reintroduction by destruction of polioviruses except in an absolute minimum number of facilities that serve essential functions and implement effective containment. Effective containment begins with a complete facility risk assessment. This article focuses on characterizing the risks of exposure to polioviruses from the essential vaccine production, quality control, and international reference and research facilities that remain. We consider the potential exposure pathways that might lead to a poliovirus reintroduction, including para-occupational exposures and releases to the environment, and review the literature to provide available estimates and a qualitative assessment of containment risks. Minimizing the risk of poliovirus transmission from a poliovirus facility to increasingly susceptible communities is a crucial and ongoing effort requiring understanding and actively managing the potential exposure pathways.

International audience; Within the past 4 years, poliomyelitis outbreaks associated with circulating vaccine-derived polioviruses (cVDPVs) have occurred in Hispaniola (2000-01), the Philippines (2001), and Madagascar (2001-02). Retrospective studies have also detected the circulation of endemic cVDPV in Egypt (1988-93) and the likely localized spread of oral poliovirus vaccine (OPV)-derived virus in Belarus (1965-66). Gaps in OPV coverage and the previous eradication of the corresponding serotype of indigenous wild poliovirus were the critical risk factors for all cVDPV outbreaks. The cVDPV outbreaks were stopped by mass immunization campaigns using OPV. To increase sensitivity for detecting vaccine-derived polioviruses (VDPVs), in 2001 the Global Polio Laboratory Network implemented additional testing requirements for all poliovirus isolates under investigation. This approach quickly led to the recognition of the Philippines and Madagascar cVDPV outbreaks, but of no other current outbreaks. The potential risk of cVDPV emergence has increased dramatically in recent years as wild poliovirus circulation has ceased in most of the world. The risk appears highest for the type 2 OPV strain because of its greater tendency to spread to contacts. The emergence of cVDPVs underscores the critical importance of eliminating the last pockets of wild poliovirus circulation, maintaining universally high levels of polio vaccine coverage, stopping OPV use as soon as it is safely possible to do so, and continuing sensitive poliovirus surveillance into the foreseeable future. Particular attention must be given to areas where the risks for wild poliovirus circulation have been highest, and where the highest rates of polio vaccine coverage must be maintained to suppress cVDPV emergence.

Nuclear mtDNA sequences (numts) are a widespread family of paralogs evolving as pseudogenes in chromosomal DNA [Zhang, D. E. & Hewitt, G. M. (1996) TREE 11, 247–251 and Bensasson, D., Zhang, D., Hartl, D. L. & Hewitt, G. M. (2001) TREE 16, 314–321]. When trying to identify the species origin of an unknown DNA sample by way of an mtDNA locus, PCR may amplify both mtDNA and numts. Indeed, occasionally numts dominate confounding attempts at species identification [Bensasson, D., Zhang, D. X. & Hewitt, G. M. (2000) Mol. Biol. Evol. 17, 406–415; Wallace, D. C., et al. (1997) Proc. Natl. Acad. Sci. USA 94, 14900–14905]. Rhesus and cynomolgus macaque mtDNA haplotypes were identified in a study of oral polio vaccine samples dating from the late 1950s [Blancou, P., et al. (2001) Nature ( London ) 410, 1045–1046]. They were accompanied by a number of putative numts. To confirm that these putative numts were of macaque origin, a library of numts corresponding to a small segment of 12S rDNA locus has been made by using DNA from a Chinese rhesus macaque. A broad distribution was found with up to 30% sequence variation. Phylogenetic analysis showed that the evolutionary trajectories of numts and bona fide mtDNA haplotypes do not overlap with the signal exception of the host species; mtDNA fragments are continually crossing over into the germ line. In the case of divergent mtDNA sequences from old oral polio vaccine samples [Blancou, P., et al. (2001) Nature ( London ) 410, 1045–1046], all were closely related to numts in the Chinese macaque library.

A search through the archives clears early vaccines of starting the AIDS pandemic. It has been suggested that chimpanzee kidney cultures may have been used in the preparation of oral polio vaccine stocks used in Africa during the late 1950s, and so could have introduced the primate precursor of the immunodeficiency virus HIV-1 into humans1,2. Here we analyse frozen samples of the suspect vaccine by using the polymerase chain reaction (PCR) to amplify any HIV-1-related nucleic acids or chimpanzee mitochondrial DNA that might be present, but we have failed to detect either. Our findings do not support the hypothesis that HIV-1 was introduced by oral vaccination against poliovirus.

Between October 2001 and April 2002, five cases of acute flaccid paralysis (AFP) associated with type 2 vaccine-derived polioviruses (VDPVs) were reported in the southern province of the Republic of Madagascar. To determine viral factors that favor the emergence of these pathogenic VDPVs, we analyzed in detail their genomic and phenotypic characteristics and compared them with co-circulating enteroviruses. These VDPVs appeared to belong to two independent recombinant lineages with sequences from the type 2 strain of the oral poliovaccine (OPV) in the 5′-half of the genome and sequences derived from unidentified species C enteroviruses (HEV-C) in the 3′-half. VDPV strains showed characteristics similar to those of wild neurovirulent viruses including neurovirulence in poliovirus-receptor transgenic mice. We looked for other VDPVs and for circulating enteroviruses in 316 stools collected from healthy children living in the small area where most of the AFP cases occurred. We found vaccine PVs, two VDPVs similar to those found in AFP cases, some echoviruses, and above all, many serotypes of coxsackie A viruses belonging to HEV-C, with substantial genetic diversity. Several coxsackie viruses A17 and A13 carried nucleotide sequences closely related to the 2C and the 3Dpol coding regions of the VDPVs, respectively. There was also evidence of multiple genetic recombination events among the HEV-C resulting in numerous recombinant genotypes. This indicates that co-circulation of HEV-C and OPV strains is associated with evolution by recombination, resulting in unexpectedly extensive viral diversity in small human populations in some tropical regions. This probably contributed to the emergence of recombinant VDPVs. These findings give further insight into viral ecosystems and the evolutionary processes that shape viral biodiversity., Author Summary Following extensive vaccination campaigns using the attenuated oral polio vaccine, wild polioviruses remain endemic in only a few countries. Nevertheless, several poliomyelitis outbreaks associated with vaccine-derived polioviruses (VDPVs) were reported in different parts of the world in recent years, particularly in Madagascar in 2002. We analyzed the molecular characteristics of Madagascar VDPVs and compared them with those of co-circulating enteroviruses. These VDPVs appear to be recombinant viruses between vaccine polioviruses and human enteroviruses of species C (HEV-C) and to present phenotypic characteristics similar to those of wild polioviruses including pathogenicity. Similar VDPVs and other enteroviruses, including several HEV-C of different types, were found in the stools of healthy children living in neighboring villages to where most of the poliomyelitis cases occurred. Some HEV-Cs showed sequences closely related to those of VDPVs, indicating genetic recombination between these viruses and vaccine polioviruses. There was also evidence of multiple genetic recombination events among other HEV-C isolates resulting in numerous different genotypes. These findings indicate that co-circulation of HEV-C and vaccine polioviruses and their evolution by recombination results in unexpectedly extensive viral diversity, at least in some small human populations, probably contributing to the emergence of recombinant VDPVs. Results of this study give further insight into the world of viruses and their biodiversity.