Circulation of a type 1 recombinant vaccine-derived poliovirus strain in a limited area in Romania.

After intensive immunisation campaigns with the oral polio vaccine (OPV) as part of the Global Polio Eradication Initiative, poliomyelitis due to wild viruses has disappeared from most parts of the world, including Europe. Here, we report the characterization of a serotype 1 vaccine-derived poliovirus (VDPV) isolated from one acute flaccid paralysis (AFP) case with tetraplegia and eight healthy contacts belonging to the same small socio-cultural group having a low vaccine coverage living in a small town in Romania. The genomes of the isolated strains appeared to be tripartite type 1/type 2/type 1 vaccine intertypic recombinant genomes derived from a common ancestor strain. The presence of 1.2% nucleotide substitutions in the VP1 capsid protein coding region of most of the strains indicated a circulation time of about 14 months. These VDPVs were thermoresistant and, in transgenic mice expressing the human poliovirus receptor, appeared to have lost the attenuated phenotype. These results suggest that small populations with low vaccine coverage living in globally well-vaccinated countries can be the origin of VDPV emergence and circulation. These results reaffirm the importance of active surveillance for acute flaccid paralysis and poliovirus in both polio-free and polio-endemic countries.

The molecular characterization of poliovirus strains by the RT-PCR-RFLP assay and its use in the active surveillance for acute flaccid paralysis cases in Romania between 2001-2006.

Poliomyelitis, an acute disease of the the central nervous system can be controlled through the use of inactivated virus vaccine (IPV) or live attenuated vaccine (OPV). The goal of the Global Polio Eradication Initiative is to stop the global transmission of poliovirus. Our study is a retrospective and prospective study that was made because in 2002 was isolated from one acute flaccid paralysis AFP case and eight healthy contacts belonging to the same small socio-cultural group having a low vaccine coverage living in Babadag a town in Romania a serotype 1 vaccine-derived poliovirus (VDPV), recombinant S1/S2/S1. The 67 poliovirus strains isolated in Romania between 2001-2006 from acute flaccid paralysis cases (AFP) (n=20, age = 3 months - 10 years), facial paralysis cases (FP) (n=5, age = 3 months - 3.7 years) and healthy's AFP contacts (n=42, age = 2 months - 5.10 years) were molecular investigated to confirm the vaccine origin of these strains and for the detection of recombinant strains. The identification of these strains was achieved through reverse transcription (RT), polymerase chain reaction (PCR) and restriction fragment length polymorphisme assays (RFLP) applied to two sequences of the viral genome, which are located in VP1-2A (2870 nt - 3648 nt) and 3D (6086nt - 6516 nt) regions. For the strains investigated in VP1-2A region, the RFLP profils after digestion with 3 restriction enzymes (Rsal, Ddel, Hinf l) were Sabin-like only with one exception, VDPV strain. In 3D region after digestion with one, two or three enzymes the genomes of most poliovirus (PV) strains were found to be similar to the original vaccine Sabin strain. In 9 AFP cases the profils detected after RFLP in 3D region were S3/S1 (n=3); S3/52 (n=2); S2/S1 (n=2), S2/S1 + S2 (n=2). In 3 FP cases the profils were S3/S2 (n=2), 53/S1 (n=1). In 11 healthy contacts the RFLP profiles in 3D region were S3/S2 (n=6); S1/S1 +S3 (n=1); S3/S1 + S2 (n=1), S3/S2 + S3 (n=1), S2 + S3/S1 (n=1), S1/S2 (n=1).

Genomic features of intertypic recombinant sabin poliovirus strains excreted by primary vaccinees.

The trivalent oral poliomyelitis vaccine (OPV) contains three different poliovirus serotypes. It use therefore creates particularly favorable conditions for mixed infection of gut cells, and indeed intertypic vaccine-derived recombinants (VdRec) have been frequently found in patients with vaccine-associated paralytic poliomyelitis. Nevertheless, there have not been extensive searches for VdRec in healthy vaccinees following immunization with OPV. To determine the incidence of VdRec and their excretion kinetics in primary vaccinees, and to establish the general genomic features of the corresponding recombinant genomes, we characterized poliovirus isolates excreted by vaccinees following primary immunization with OPV. Isolates were collected from 67 children 2 to 60 days following vaccination. Recombinant strains were identified by multiple restriction fragment length polymorphism assays. The localization of junction sites in recombinant genomes was also determined. VdRec excreted by vaccinees were first detected 2 to 4 days after vaccination. The highest rate of recombinants was on day 14. The frequency of VdRec depends strongly on the serotype of the analyzed isolates (2, 53, and 79% of recombinant strains in the last-excreted type 1, 2, and 3 isolates, respectively). Particular associations of genomic segments were preferred in the recombinant genomes, and recombination junctions were found in the genomic region encoding the nonstructural proteins. Recombination junctions generally clustered in particular subgenomic regions that were dependent on the serotype of the isolate and/or on the associations of genomic segments in recombinants. Thus, VdRec are frequently excreted by vaccinees, and the poliovirus replication machinery requirements or selection factors appear to act in vivo to shape the features of the recombinant genomes.

Natural genetic exchanges between vaccine and wild poliovirus strains in humans.

In a previous study of poliovirus vaccine-derived strains isolated from patients with vaccine-associated paralytic poliomyelitis (VAPP) (9, 11), we reported that a high proportion (over 50%) of viruses had a recombinant genome. Most were intertypic vaccine/vaccine recombinants. However, some had restriction fragment length polymorphism (RFLP) profiles different from those of poliovirus vaccine strains. We demonstrate here that five such recombinants, of 88 VAPP strains examined, carried sequences of wild (nonvaccine) origin. To identify the parental wild donor of these sequences, we used RFLP profiles and nucleotide sequencing to look for similarity in the 3D polymerase-coding region of 61 wild, cocirculating poliovirus isolates (43 type 1, 16 type 2, and 2 type 3 isolates). In only one case was the donor identified, and it was a wild type 1 poliovirus. For the other four vaccine/wild recombinants, the wild parent could not be identified. The possibility that the wild sequences were of a non-poliovirus-enterovirus origin could not be excluded. Another vaccine/wild recombinant, isolated in Belarus from a VAPP case, indicated that the poliovirus vaccine/wild recombination is not an isolated phenomenon. We also found wild polioviruses (2 of 15) carrying vaccine-derived sequences in the 3' moiety of their genome. All these results suggest that genetic exchanges with wild poliovirus and perhaps with nonpoliovirus enteroviruses, are also a natural means of evolution for poliovirus vaccine strains.

Evolution of the Sabin type 1 poliovirus in humans: characterization of strains isolated from patients with vaccine-associated paralytic poliomyelitis.

Attenuated strains of the Sabin oral poliovirus vaccine replicate in the human gut and in rare cases cause vaccine-associated paralytic poliomyelitis (VAPP). Reversion of vaccine strains toward a pathogenic phenotype is probably one of the main causes of VAPP, a disease most frequently associated with type 3 and type 2 strains and more rarely with the type 1 (Sabin 1) strain. To identify the determinants and mechanisms of safety versus pathogenicity of the Sabin 1 strain, we characterized the genetic and phenotypic changes in six Sabin 1-derived viruses isolated from immunocompetent patients with VAPP. The genomes of these strains carried either few or numerous mutations from the original Sabin 1 genome. As assessed in transgenic mice carrying the human poliovirus receptor (PVR-Tg mice), all but one strain had lost the attenuated phenotype. Four strains presented only a moderate neurovirulent phenotype, probably due at least in part to reversions to the wild-type genotype, which were detected in the 5' noncoding region of the genome. The reversions found in most strains at nucleotide position 480, are known to be associated with an increase in neurovirulence. The construction and characterization of Sabin 1 mutants implicated a reversion at position 189, found in one strain, in the phenotypic change. The presence of 71 mutations in one neurovirulent strain suggests that a vaccine-derived strain can survive for a long time in humans. Surprisingly, none of the strains analyzed were as neurovirulent to PVR-Tg mice as was the wild-type parent of Sabin 1 (Mahoney) or a previously identified neurovirulent Sabin 1 mutant selected at a high temperature in cultured cells. Thus, in the human gut, the Sabin 1 strain does not necessarily evolve toward the genetic characteristics and high neuropathogenicity of its wild-type parent.

Sequential and combined use of inactivated and oral poliovirus vaccines: Dolj District, Romania, 1992-1994.

To determine the feasibility of a vaccination strategy that would reduce the risk of vaccine-associated paralysis while retaining a barrier against the spread of wild poliovirus, a 2-year project was undertaken using enhanced-potency inactivated poliovirus vaccine (IPV) administered at 2 and 3 months of age followed by doses of both IPV and oral poliovirus vaccine (OPV) administered at 4 and 9 months of age. Vaccination coverage by 12 months of age with three or more doses of IPV and two doses of OPV among 16,566 infants eligible for vaccination was > 95% and > 80%, respectively. Among 51 children from whom blood samples were obtained 45 days after their third dose of IPV and first dose of OPV, 100% had serum neutralizing antibodies (reciprocal titer > or = 10) to all three poliovirus types. No cases of paralytic poliomyelitis due to either wild or vaccine-related strains were reported. The project demonstrated the feasibility, safety, and high immunogenicity of sequential use of IPV followed by OPV in Romania.

Neutralization epitope patterns of poliovirus strains isolated from cases of acute spinal paralysis and from healthy vaccinated children.

By using a battery of poliovirus neutralizing monoclonal antibodies received from the Pasteur Institute, Paris, we carried out the study of the patterns of neutralization epitopes of more than 300 poliovirus strains isolated from cases of acute spinal paralysis and from healthy vaccinated children. We tried to find out the utility of this test for establishing the origin (wild or Sabin) of a poliovirus strain and the possible evolution of the pattern of neutralization epitopes. The study of the neutralization epitopes was carried out by neutralization index assay (NI). The neutralization index represents the difference between the titers of poliovirus strain determined in parallel in the absence, as well as in the presence of constant concentration (predetermined) for each monoclonal antibody. An epitope was considered to be present for a NI > or = 2 and absent for a NI < or = 1.5. For types 1 and 3 polioviruses, Sabin-like epitopes have never been found to be present simultaneously with wild type epitopes on the same virus particle. Therefore, examination of the epitope pattern of these strains is very useful for intratypic differentiation. On the other hand, in type 2 poliovirus strains, Sabin and wild type epitopes sometimes coexist, in various combinations, thus rendering the interpretation of the results rather difficult.

Paralytic poliomyelitis in Romania, 1984-1992. Evidence for a high risk of vaccine-associated disease and reintroduction of wild-virus infection.

Although poliomyelitis due to wild-virus infection has virtually disappeared from Romania, with no cases having been documented between 1984 and 1989, vaccine-associated paralytic poliomyelitis has been reported at very high rates for over two decades. In November 1990, to decrease the risk of vaccine-associated paralytic poliomyelitis, oral poliovirus vaccine produced in Romania was replaced by imported oral vaccine made by a Western European manufacturer. To better quantify the risk of vaccine-associated paralytic poliomyelitis and the impact of the change in vaccine manufacturer, the authors reviewed clinical, epidemiologic, and laboratory data on poliomyelitis cases that occurred in Romania from 1984 to 1992. Poliovirus isolates were characterized at the US Centers for Disease Control and Prevention. During the period 1984-1992, 132 confirmed cases of paralytic poliomyelitis were reported in Romania, of which 13 were classified as wild-virus-associated, 93 as vaccine-associated, and 26 as "of unknown origin." Wild type 1 poliovirus was isolated during 1990-1992 from nine of 13 (69%) cases in an outbreak that occurred primarily among undervaccinated gypsy children. Vaccine-associated cases were epidemiologically and virologically distinct from wild-virus cases. Of the 93 vaccine-associated cases, 45 children were recipients and 48 were contacts. The overall risk of vaccine-associated paralytic poliomyelitis in Romania (1 case per 183,000 doses of oral poliovirus vaccine distributed) was 14-fold higher than the risk in the United States. The risks of recipient vaccine-associated paralytic poliomyelitis related to the first dose of oral vaccine were similar for Romanian and imported vaccine (1 case per 95,000 doses and 1 case per 65,000 doses, respectively), as were the total risks of vaccine-associated paralytic poliomyelitis. These findings definitively demonstrate a substantially elevated risk of vaccine-associated paralytic poliomyelitis in Romania which was not affected by a change in oral poliovirus vaccine manufacturer.

High diversity of poliovirus strains isolated from the central nervous system from patients with vaccine-associated paralytic poliomyelitis.

To establish the etiology of vaccine-associated paralytic poliomyelitis (VAPP), isolates from the central nervous system (CNS) from eight patients with VAPP were compared with stool isolates from the same patients. The vaccine (Sabin) origin was checked for all of the available isolates. Unique and similar strains were recovered from paired stool and CNS samples for five of the eight VAPP cases and the three wild-type cases included in the study. In the remaining three VAPP cases, the stool samples and, in one case, the CNS samples contained mixtures of strains. In two of these cases an equivalent of the CNS isolate was found among the strains separated by plaque purification from stool mixtures, and in one case different strains were isolated from CNS and stool. This shows that the stool isolate in VAPP might not be always representative of the etiologic agent of the neurological disease. A wide variety of poliovirus vaccine genomic structures appeared to be implicated in the etiology of VAPP. Of nine CNS vaccine-derived strains, four were nonrecombinant and five were recombinant (vaccine/vaccine or even vaccine/nonvaccine). The neuropathogenic potential of the isolates was evaluated in transgenic mice sensitive to poliovirus. All of the CNS-isolated strains lost the attenuated phenotype of the Sabin strains. However, for half of them, the neurovirulence was lower than expected, suggesting that the degree of neurovirulence for transgenic mice is not necessarily correlated with the neuropathogenicity in humans.