Use of a multiple restriction fragment length polymorphism method for detecting vaccine-derived polioviruses in clinical samples.

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.

New PCR test that recognizes all human prototypes of enterovirus: application for clinical diagnosis.

We describe a new PCR test (Penter RT-PCR) that recognizes all 64 prototypes of enterovirus. Sixty clinical samples were analyzed in parallel with this Penter RT-PCR and previously described PCR tests: 34 and 32 samples tested positive, respectively. This assay is suitable for use in clinical diagnosis, and its ability to amplify all known serotypes makes it more useful than other consensus PCR tests.

Genetic recombination in wild-type poliovirus.

Poliovirus isolates were screened for recombinants by combined analysis of two distant polymorphic segments of the poliovirus genome (one in the capsid and the other in the polymerase-coding region). Using a restriction fragment length polymorphism (RFLP) assay, a high number of recombinant genomes was found among vaccine-derived strains excreted by poliovirus vaccine vaccinees or vaccine-associated paralytic poliomyelitis cases. Some of these subjects carried a wild-type poliovirus (non-vaccine-specific) nucleotide sequence in the 3' part of the genome.

Natural genetic recombination between co-circulating heterotypic enteroviruses.

Natural recombination in poliovirus is a frequent phenomenon. In practice, whenever different genotypes have the opportunity to infect the same individual, a high proportion of viruses with recombinant genomes are excreted. To determine whether enteroviruses other than poliovirus can naturally produce viable virions with recombinant genomes, we studied the molecular features of two distant regions of the viral genomes - the VP1 coding region and the 3D polymerase coding region - of the echovirus serotypes associated with a large outbreak of aseptic meningitis.

Molecular strategy for 'serotyping' of human enteroviruses.

To explore further the phylogenetic relationships between human enteroviruses and to develop new diagnostic approaches, we designed a pair of generic primers in order to study a 1452 bp genomic fragment (relative to the poliovirus Mahoney genome), including the 3' end of the VP1-coding region, the 2A- and 2B-coding regions, and the 5' moiety of the 2C-coding region. Fifty-nine of the 64 prototype strains and 45 field isolates of various origins, involving 21 serotypes and 6 strains untypable by standard immunological techniques, were successfully amplified with these primers. By determining the nucleotide sequence of the genomic fragment encoding the C-terminal third of the VP1 capsid protein we developed a molecular typing method based on RT-PCR and sequencing.