Molecular Properties of Poliovirus Isolates: Nucleotide Sequence Analysis, Typing by PCR and Real-Time RT-PCR

Abstract

Virologic surveillance is essential to the success of the World Health Organization initiative to eradicate poliomyelitis. Molecular methods have been used to detect polioviruses in tissue culture isolates derived from stool samples obtained through surveillance for acute flaccid paralysis. This chapter describes the use of realtime PCR assays to identify and serotype polioviruses. In particular, a degenerate, inosine-containing, panpoliovirus (panPV) PCR primer set is used to distinguish polioviruses from NPEVs. The high degree of nucleotide sequence diversity among polioviruses presents a challenge to the systematic design of nucleic acid-based reagents. To accommodate the wide variability and rapid evolution of poliovirus genomes, degenerate codon positions on the template were matched to mixed-base or deoxyinosine residues on both the primers and the TaqMan™ probes. Additional assays distinguish between Sabin vaccine strains and non-Sabin strains. This chapter also describes the use of generic poliovirus specific primers, along with degenerate and inosine-containing primers, for routine VP1 sequencing of poliovirus isolates. These primers, along with nondegenerate serotype-specific Sabin primers, can also be used to sequence individual polioviruses in mixtures.

Update on Vaccine-Derived Polioviruses — Worldwide, January 2016–June 2017

“…All poliovirus isolates are characterized by laboratories of the Global Polio Laboratory Network. VDPV screening is conducted using real-time reverse transcription–polymerase chain reaction (RT-PCR) nucleic acid amplification, targeted to nucleotide substitutions that frequently revert to the parental WPV sequence during replication of OPV in the human intestine ( 6 ). Starting August 1, 2016, the use of the VDPV2 screening assay was discontinued and all PV2 isolates are sequenced.…”

Diagnostic Assay Development for Poliovirus Eradication

“…With the introduction of the PV2 assay, the testing algorithm was changed and the VDPV2 assay became obsolete, since all type 2 viruses must now be referred for sequencing ( Fig. S4) (9,24). The design of the ITD 5.0 algorithm incorporates redundancies to detect all polioviruses with multiple assays to protect against genetic drift or user error.…”

“…Regardless of its reduced sensitivity, the PanPV assay is a cornerstone of the ITD 5.0 algorithm, since it captures all poliovirus serotypes and genotypes. Following the ITD 5.0 algorithm, these viruses would still be detected as indeterminate polioviruses (positive for PanPV and PanEV but negative in all other assays or positive for PanEV, negative for PanPV, but positive for one or more of the other assays) and would be referred for VP1 sequencing (24).…”

“…cDNAs were synthesized with reverse-sense primers 7500A and Q8, and the remaining single-stranded RNA was digested with RNase H at 37°C for 30 min. The two cDNA fragments were amplified using a combination of primers 28S and Q8 (5= amplicon) or Y7 and 7500A (3= amplicon) (see Table S4 in the supplemental material) (24)(25)(26). PCR amplification using Q5 high-fidelity polymerase was run at 98°C for 30 s, followed by 35 cycles of 98°C for 10 s, 50°C for 30 s, and 72°C for 3 min, with a final incubation at 72°C for 5 min (New England BioLabs, Ipswich, MA).…”

Acute flaccid paralysis surveillance in bosnia and herzegovina: Recent isolation of two sabin like type 2 poliovirus

“…The Sabin‐like isolates were also analyzed by rRT‐PCR VDPV assay in order to identify VDVP. Finally, VP1 region of RNA extracted from poliovirus isolates, was amplified by RT‐PCR and sequenced . Nucleotide sequences were aligned using Sequencer software (Gene Codes Corporation Ann Arbor, Michigan) and compared with the reference type sequences to identify nucleotide substitutions and percentage of identity.…”