Serotype-specific identification of polioviruses by PCR using primers containing mixed-base or deoxyinosine residues at positions of codon degeneracy

Abstract

We have developed a method for determining the serotypes of poliovirus isolates by PCR. Three sets of serotype-specific antisense PCR-initiating primers (primers seroPV1A, seroPV2A, and seroPV3A) were designed to pair with codons of VP1 amino acid sequences that are conserved within but that differ across serotypes. The sense polarity primers (primers seroPV1S, seroPV2S, and seroPV3S) matched codons of more conserved capsid sequences. The primers contain mixed-base and deoxyinosine residues to compensate for the high rate of degeneracy of the targeted codons. The serotypes of all polioviruses tested (48 vaccine-related isolates and 110 diverse wild isolates) were correctly identified by PCR with the serotype-specific primers. None of the genomic sequences of 49 nonpolio enterovirus reference strains were amplified under equivalent reaction conditions with any of the three primer sets. These primers are useful for the rapid screening of poliovirus isolates and for determining the compositions of cultures containing mixtures of poliovirus serotypes.

FIG. 1

The locations along poliovirus genome of the sequences targeted by seroPV PCR primers are depicted in the diagram at the top. Untranslated regions are indicated as lines; the region of the translated polyprotein is represented by the rectangle. Shaded areas indicate locations of surface loops forming neutralization antigenic sites 1, 2a, and 3 (20). Arrows indicate the locations and polarities of the seroPV PCR primers. The alignment of the amino acid residues (in boldface type) whose codons are specifically bound by the seroPV PCR primers is indicated below. The surface residues forming neutralizing antigenic site 2a (20) are underlined. Amino acid differences among the target sites within and across serotypes are shown for 20 independent poliovirus isolates (13). The locations of capsid amino acid residues are given by four-digit numbers (15): the first digit identifies the virion protein and the next three digits specify residue position (e.g., 1001 indicates residue 1 of VP1). Country abbreviations are as defined by the World Health Organization (5).

FIG. 2

Alignment of seroPV PCR primers with poliovirus target sequences. The targeted amino acid sequences (top) are aligned with all possible encoding nucleotide sequences (middle) and the sequences of the seroPV PCR primers (bottom). Abbreviations for nucleotides follow the International Union of Biochemistry nomenclature (22): H, adenine, cytosine, or thymine; I, inosine; N, adenine, cytosine, guanine, or thymine; R, adenine or guanine; Y, cytosine or thymine.

FIG. 3

Specificity of PCR amplification with the seroPV1 PCR primers, which yields a 70-bp product. Products were visualized after polyacrylamide gel electrophoresis by ethidium bromide fluorescence as described previously (30). (A to C) Lanes 1, amplicon from Sabin 1 template (positive control); lanes N, negative template control; lanes M, molecular weight marker V (57 to 587 bp) from Boehringer Mannheim; lanes 2 to 13, amplification products obtained with templates of different wild poliovirus isolates. (A) Poliovirus type 1. Lanes 2, 6070/CHN94; 3, 5558/NIE94; 4, 5386/ANG94; 5, 5058/THA93; 6, 3894/EGY92; 7, 3866/IND92; 8, 3862/TOG92; 9, 3647/CHN91; 10, 2781/VTN91; 11, 0467/COL91; 12, 0919/GEO90; and 13, 9188/BRA88. (B) Poliovirus type 2. Lanes 2, 3874/IND92; 3, 3825/PAK91; 4, 3845/EGY91; 5, 2613/PAK89; 6, 0176/PER89; 7, 7079/IND86; 8, 1534/IND82; 9, 0301/CAE80; 10, 0298/EGY79; 11, 0298/ISR78; 12, MEF-1/EGY42; and 13, Lansing/USA37. (C) Poliovirus type 3. Lanes: 2, 6071/CHN93; 3, 5376/PHL93; 4, 3899/EGY92; 5, 3873/IND92; 6, 0780/OMA91; 7, 3997/TKM90; 8, 2723/TUR90; 9, 9288/MEX89; 10, 9035/BRA88; 11, 8854/COL88; 12, 23127/FIN84; and 13, Saukett/USA52.

FIG. 4

PCR amplification with the seroPV2 PCR primers, which yields a 79-bp product. (A to C) Lanes 1, amplicon from Sabin 2 template. Samples in all other lanes are as described in the legend to Fig. 3.

FIG. 5

PCR amplification with the seroPV3 PCR primers, which yields a 140-bp product. (A to C) Lanes 1, amplicon from Sabin 3 template. Samples in all other lanes are as described in the legend to Fig. 3.