Serial recombination during circulation of type 1 wild-vaccine recombinant polioviruses in China

Abstract

Type 1 wild-vaccine recombinant polioviruses sharing a 367-nucleotide (nt) block of Sabin 1-derived sequence spanning the VP1 and 2A genes circulated widely in China from 1991 to 1993. We surveyed the sequence relationships among 34 wild-vaccine recombinants by comparing six genomic intervals: the conserved 5'-untranslated region (5'-UTR) (nt 186 to 639), the hypervariable portion of the 5'-UTR (nt 640 to 742), the VP4 and partial VP2 genes (nt 743 to 1176), the VP1 gene (nt 2480 to 3385), the 2A gene (nt 3386 to 3832), and the partial 3D gene (nt 6011 to 6544). The 5'-UTR, capsid (VP4-VP2 and VP1), and 2A sequence intervals had similar phylogenies. By contrast, the partial 3D sequences could be distributed into five divergent genetic classes. Most (25 of 34) of the wild-vaccine recombinant isolates showed no evidence of additional recombination beyond the initial wild-Sabin recombination event. Eight isolates from 1992 to 1993, however, appear to be derived from three independent additional recombination events, and one 1993 isolate was derived from two consecutive events. Complete genomic sequences of a representative isolate for each 3D sequence class demonstrated that these exchanges had occurred in the 2B, 2C, and 3D genes. The 3D gene sequences were not closely related to those of the Sabin strains or 53 diverse contemporary wild poliovirus isolates from China, but all were related to the 3D genes of species C enteroviruses. The appearance within approximately 2.5 years of five recombinant classes derived from a single ancestral infection illustrates the rapid emergence of new recombinants among circulating wild polioviruses.

FIG. 1.

Nucleotide similarity plots comparing the complete genomic sequences of an early poliovirus type 1 wild-vaccine recombinant isolate Hebei/91-2 (recombinant class R91-1) with those of a closely related type 1 wild isolate Henan/91-3 (genotype JX89) (top graph) and with those of the Sabin type 1 OPV strain (bottom graph). The plots are aligned with a schematic of the organization of the poliovirus genome (top); the single open reading frame is flanked by the 5′- and 3′-UTRs.

FIG. 2.

Maximum-likelihood trees of 34 wild-vaccine recombinant poliovirus isolates from China, constructed from nucleotide sequences of six genomic intervals. (A) 5′-UTR conserved region (nt 186 to 639); (B) 5′-UTR hypervariable region (nt 640 to 742); (C) complete VP4 and partial VP2 gene interval (nt 743 to 1176); (D) complete VP1 gene (nt 2480 to 3385); (E) complete 2A gene (nt 3386 to 3832); F, partial 3D gene sequence interval (nt 6011 to 6544). Each tree was rooted to the sequence of Hebei/91-2. Brackets, lineages (A to G) and sublineages (e.g., C1 and C2), labeled as previously described for the VP1 and 2A gene regions (23). Isolates representing each class of new recombinant 3D gene sequences are identified by symbols (R91-2, solid square; R91-3, solid circle; R91-4, open triangle; R91-5, solid triangle).

FIG. 3.

Mapping of recombination junction regions in representatives of each of the five R91 3D gene recombinant classes. (A) Nucleotide similarity plots comparing the complete genomic sequences of poliovirus type 1 wild-vaccine isolates representing each recombinant class: R91-1, Hebei/91-2; R91-2, Yunnan/92; R91-3, Guangdong/92-2; R91-4, Hainan/93-2; R91-5, Fujian/93-8. The similarity plots are aligned with a schematic of the poliovirus genome. The four intervals below the schematic (a, nt 1 to 4008; b, nt 4009 to 4842; c, nt 4843 to 6378; d, nt 6379 to 7441) correspond to genomic intervals bounded by recombination sites. The reference sequence in four plots is the early recombinant isolate Hebei/91-2, and the location of the crossover site in the sequence of the later recombinant isolate is in parentheses. Sequences of the closely related cluster C2 isolates Guangdong/92-2 and Hainan/93-2 were also directly compared. (B) Nucleotide sequence alignments of the sequences surrounding the predicted recombination sites in the nonstructural protein genes of wild-vaccine recombinant polioviruses. Newly incorporated nucleotide sequences derived from unknown sources are shaded and define the right (downstream) boundaries of crossover sites. Darker shade indicates nucleotide sequence derived from consecutive recombination in the 2C gene in group R91-4. (C) Unrooted maximum-likelihood trees summarizing sequence relationships across four genomic intervals bounded by recombination sites. The interval between the two recombination sites in the 2C gene was not included because it is too short for meaningful phylogenetic analysis. The input Ts/Tv ratios were estimated from program TREE-PUZZLE, version 5.0, to be 7.50, 3.40, 3.30, and 2.70 for the four intervals. The trees are plotted to the same scale, indicated by the scale bar at lower left.

FIG. 4.

Approximate location of crossover sites of the five different classes of wild-vaccine recombinant polioviruses found in China from 1991 to 1993. Black bars, sequences derived from the indigenous type 1 wild poliovirus genotype JX89; white bars, Sabin 1-derived sequences; other patterns, sequences derived from enteroviruses other than the Sabin OPV strains. The bars symbolize the derivation of sequences of the wild parent, Henan/91-3 (genotype JX89), and representatives of each of the five 3D recombinant classes: R91-1, Hebei/91-2; R91-2, Yunnan/92; R91-3, Guangdong/92-2; R91-4, Hainan/93-2; R91-5, Fujian/93-8.

FIG. 5.

Unrooted maximum-likelihood tree summarizing complete 3D gene sequence relationships among poliovirus and NPEV isolates and reference strains. In addition to the 3D gene sequences of the nonrecombinant JX89 isolate Henan/91-2 and of representative isolates of wild-vaccine recombinant classes R91-1 to R91-5 determined in this study, 3D gene sequences of the following reference strains were obtained from GenBank (accession numbers in parentheses): coxsackievirus A1 (CAV1; AF499635), CAV9 (D00627), CAV11 (AF499636), CAV13 (AF499637), CAV15 (AF499638), CAV16 (NC001612), CAV17 (AF499639), CAV18 (AF499640), CAV19 (AF499641), CAV20 (AF499642), CAV21 (D00538), CAV22 (AF499643), CAV24 (D90457), CBV1 (M16560), CBV2 (AF085363), CBV3 (M88483), CBV4 (X05690), CBV5 (X67706), CBV6 (AF105342), echovirus 5 (ECV5; AF083069), ECV9 (X92886), ECV11 (X80059), ECV12 (X79047), enterovirus 70 (EV70; D00820), EV71 (U22521), EV71 (China strain SHZH98; AF302996), Sabin 1 (Sab 1; J02285), Sab 2 (X00595), and Sab 3 (X00596).

FIG. 6.

Geographic distribution of the 34 type 1 wild-vaccine poliovirus recombinant isolates that evolved to five different recombinant classes in China from 1991 to 1993. Province abbreviations: AN, Anhui; FJ, Fujian; GD, Guangdong; GS, Gansu; GX, Guangxi; GZ, Guizhou; HA, Hainan; HB, Hebei; HE, Henan; HL, Heilongjiang; HN, Hunan; HU, Hubei; JL, Jilin; JS, Jiangsu; JX, Jiangxi; LN, Liaoning; NM, Nei Mongol; NX, Ningxia; QH, Qinghai; SA, Shaanxi; SC, Sichuan; SD, Shandong; SX, Shanxi; XJ, Xinjiang; XZ, Xizang (Tibet); YN, Yunnan; ZJ, Zhejiang.