Development of a multiplex RT-PCR assay for the identification of recombination types at different genomic regions of vaccine-derived polioviruses.
Polioviruses (PVs) are the causal agents of acute paralytic poliomyelitis. Since the 1960s, poliomyelitis has been effectively controlled by the use of two vaccines containing all three serotypes of PVs, the inactivated poliovirus vaccine and the live attenuated oral poliovirus vaccine (OPV). Despite the success of OPV in polio eradication programme, a significant disadvantage was revealed: the emergence of vaccine-associated paralytic poliomyelitis (VAPP). VAPP is the result of accumulated mutations and putative recombination events located at the genome of attenuated vaccine Sabin strains. In the present study, ten Sabin isolates derived from OPV vaccinees and environmental samples were studied in order to identify recombination types located from VP1 to 3D genomic regions of virus genome. The experimental procedure that was followed was virus RNA extraction, reverse transcription to convert the virus genome into cDNA, PCR and multiplex-PCR using specific designed primers able to localize and identify each recombination following agarose gel electrophoresis. This multiplex RT-PCR assay allows for the immediate detection and identification of multiple recombination types located at the viral genome of OPV derivatives. After the eradication of wild PVs, the remaining sources of poliovirus infection worldwide would be the OPV derivatives. As a consequence, the immediate detection and molecular characterization of recombinant derivatives are important to avoid epidemics due to the circulation of neurovirulent viral strains.
Microbiology-Virology Laboratory, Department of Biochemistry & Biotechnology, School of Health Sciences, University of Thessaly, Ploutonos 26 & Aiolou, 41221, Larissa, Greece.
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Dimitriou
Microbiology-Virology Laboratory, Department of Biochemistry & Biotechnology, School of Health Sciences, University of Thessaly, Ploutonos 26 & Aiolou, 41221, Larissa, Greece.
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Kyriakopoulou
Microbiology-Virology Laboratory, Department of Biochemistry & Biotechnology, School of Health Sciences, University of Thessaly, Ploutonos 26 & Aiolou, 41221, Larissa, Greece.
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Tsakogiannis
Microbiology-Virology Laboratory, Department of Biochemistry & Biotechnology, School of Health Sciences, University of Thessaly, Ploutonos 26 & Aiolou, 41221, Larissa, Greece.
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Fikatas
Department of Microbiology, Medical School, University of Ioannina, Ioannina, Greece.
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Gartzonika
Department of Microbiology, Medical School, University of Ioannina, Ioannina, Greece.
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Levidiotou-Stefanou
Microbiology-Virology Laboratory, Department of Biochemistry & Biotechnology, School of Health Sciences, University of Thessaly, Ploutonos 26 & Aiolou, 41221, Larissa, Greece. markoulatos@bio.uth.gr.
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Markoulatos
D017931:DNA Primers / Q000235:genetics; D016679:Genome, Viral / Q000235:genetics; D006801:Humans; D060885:Multiplex Polymerase Chain Reaction / Q000379:methods*; D011051:Poliomyelitis / Q000276:immunology / Q000821:virology; D017955:Poliovirus / Q000235:genetics* / Q000276:immunology; D011055:Poliovirus Vaccine, Oral / Q000276:immunology; D012367:RNA, Viral / Q000235:genetics; D011995:Recombination, Genetic / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*
D017931:DNA Primers; D011055:Poliovirus Vaccine, Oral; D012367:RNA, Viral
Identification; Multiplex RT-PCR; OPV derivatives; Recombination; VAPP