Antigenic diversity of enteroviruses associated with nonpolio acute flaccid paralysis, India, 2007-2009

Abstract

Because of the broadened acute flacid paralysis (AFP) definition and enhanced surveillance, many nonpolio AFP (NP-AFP) cases have been reported in India since 2005. To determine the spectrum of nonpolio enterovirus (NPEV) serotypes associated with NP-AFP from polio-endemic and -free regions, we studied antigenic diversity of AFP-associated NPEVs. Of fecal specimens from 2,786 children with NP-AFP in 1 polio-endemic and 2 polio-free states, 823 (29.5%) were positive for NPEVs in RD cells, of which 532 (64.6%) were positive by viral protein 1 reverse transcription PCR. We identified 66 serotypes among 581 isolates, with enterovirus 71 most frequently (8.43%) detected, followed by enterovirus 13 (7.1%) and coxsackievirus B5 (5.0%). Most strains within a serotype represented new genogropups or subgenogroups. Agents for ≈35.0% and 70.0% of culture-positive and -negative cases, respectively, need to be identified. Association of human enterovirus with NP-AFP requires better assessment and understanding of health risks of NPEV infections after polio elimination.

Figure 1

Strains belonging to each serotype detected in children with acute febrile paralysis, India, 2007–2009. CA, coxsackievirus; E, echovirus; EV, enterovirus.

Figure 2

Phylogenetic tree based on viral protein 1 gene nucleotide sequences of strains representing each of the 66 enterovirus serotypes detected in children with acute febrile paralysis, India, 2007–2009. The phylogenetic tree was constructed by using the maximum-parsimony method and with search level 1 close-neighbor-interchange algorithm. The percentage of replicate trees in which the associated types clustered together in the bootstrap test (500 replicates) is shown next to the branches. Evolutionary analyses were conducted in MEGA5 (13). The viral protein 1 gene sequences of the 3 poliovirus prototypes were used as reference. The strain numbers with their serotype association are indicated. The GenBank accession numbers of VP1 gene for 618 acute febrile paralysis isolates are HQ454497–454499 and JN203499–JN204113.

Figure 3

Phylogenetic analyses of viral protein 1 sequences of enterovirus 71 and echovirus 13 strains with those of reference strains representing different genogroups and subgenogroups within a serotype, India, 2007–2009. Multiple sequence alignments were performed by using ClustalW program (www.genome.jp/tools/clustalw/) and phylogenetic analysis by MEGA5 program (13) with pairwise comparison and maximum composite likelihood nucleotide substitution model. Phylogenetic trees were constructed by UPGMA (unweighted pair group method using arithmetic averages) with statistical significance of the phylogenetic analyses estimated by bootstrap analysis with 1,000 pseudoreplicate datasets. A and B represent phylogenetic trees of viral protein 1 sequences of enterovirus 71 and echovirus 13, respectively. The serotype, state and year of isolation of each strain and GenBank accession numbers of reference strains used are indicated. 1000B is an echovirus 1 strain. Scale bars indicate nucleotide substitutions per site.

Figure 4

Phylogenetic tree of viral protein 1 sequences of coxsackievirus B1–B6 isolates generated in comparison with those of strains belonging to different genotypes of B1, B2, B3, B4, B5, and B6 serotypes, India, 2007–2009. Multiple sequence alignments were performed by using ClustalW program (www.genome.jp/tools/clustalw/) and phylogenetic analysis by MEGA5 program (13) with pairwise comparison and maximum composite likelihood nucleotide substitution model. Phylogenetic trees were constructed by UPGMA (unweighted pair group method using arithmetic averages) with statistical significance of the phylogenetic analyses estimated by bootstrap analysis with 1,000 pseudoreplicate datasets.