Complete genome analysis of coxsackievirus A2, A4, A5, and A10 strains isolated from hand, foot, and mouth disease patients in China revealing frequent recombination of human enterovirus A

Abstract

Coxsackievirus (CV) strains CVA2, CVA4, CVA5, and CVA10 were isolated from patients with hand, foot, and mouth disease during a 2009 outbreak in China. Full genome sequences for four representative strains, CVA2/SD/CHN/09 (A2SD09), CVA4/SZ/CHN/09 (A4SZ09), CVA5/SD/CHN/09 (A5SD09), and CVA10/SD/CHN/09 (A10SD09), were determined. Phylogenetic and recombination analyses of the isolates by comparison with human enterovirus A prototype strains revealed that genetic recombination occurred during cocirculation of the viruses. The A2SD09 and A4SZ09 strains were most closely related to their corresponding prototype strains in the capsid region but shared noncapsid sequences with each other. Similarly, strains A5SD09 and A10SD09 had serotype-specific homology for the capsid proteins but shared noncapsid sequences with each other. Phylogenetic analyses of the four isolates with homotypic strains showed that CVA2 strains were divided into five genotypes. The A2SD09 strain clustered with Mongolia strains isolated in 2003, forming genotype V. The A4SZ09 strain and other isolates from mainland China and Taiwan clustered with genotype III strains and are likely related to strains that circulated in Europe and Mongolia. The A5SD09 strain is closely related to other Chinese strains isolated in 2008. The A10SD09 isolate, together with other Chinese strains isolated since 2004, formed a distinct lineage that was likely imported from Japan and South Korea. This study shows that natural recombination is a frequent event in human enterovirus A evolution. More comprehensive surveillance of enteroviruses that focus not only on EV71 or CVA16 is needed for us to understand the molecular epidemiology of enteroviruses and to track recombination events which may ultimately affect the virulence of viruses during outbreaks.

Fig. 1

Maximum likelihood trees constructed on the basis of the comparisons of different regions of genomes of the four new 2009 enteroviruses and all other prototype strains and modern strains in HEV-A species by using the PhyML, version 3.0, program. (a) P1 region; (b) VP1 region; (c) P3 region; (d) 3D region. Bootstrap values (the percentage of 1,000 pseudoreplicate data sets) lower than 70% are not shown for clarity. Each of the trees includes a representative (CVB1, poliovirus, and EV70) of each of the other three human enterovirus species (HEV-B, -C, and -D) as reference points. The sequenced isolates are indicated by triangles. Sequences with the following GenBank accession numbers were used: CVA2 to CVA8, CVA10, CVA12, and CVA14, AY421760 to AY421769, respectively; CVA16G10, U05876; CVA16ShZh00, AY790926; EV71 BrCr, U22521; EV71, FY0805 and FJ439769; EV76, AY697458; EV89, AY697459; EV90, AB192877; EV90, F950027 and AY773285; EV91, AY697461; EV92, EF667344; CVB1, M16560; poliovirus, NC_002058; EV70, DQ201177.

Fig. 2

Simplot and bootscanning analyses of the four 2009 HEV-A strains and other HEV-A prototype strains on the basis of full-length genomes. Each analysis used each of the four new viruses as the query sequence. A sliding window of 400 nucleotides moving in 50-nucleotide steps was used in this analysis. (a and b) A2SD09; (c and d) A4SZ09; (e and f): A5SD09; (g and h) A10SD09.

Fig. 3

Phylogenetic dendrograms constructed by using the neighbor-joining method and the MEGA, version 4.0, program and based on the alignment of the common 3′ partial VP1 sequences of the new 2009 strains and all other corresponding strains from GenBank. The bootstrap values from 1,000 pseudoreplicates for major lineages within the tree are shown as percentages. Bootstrap values lower than 70% are not shown for clarity. The prototype CVA16 strain (G10) was used as an outgroup. The sequenced isolates are indicated by triangles. (a) A2SD09, nt 2919 to 3320; (b) A4SZ09, nt 2923 to 3250; (c) A5SD09, nt 3006 to 3273; (d) A10SD09, nt 2917 to 3301. Sequences with the following GenBank accession numbers were used: CVA2, AB162720.1, AB162722.1, DQ317258.1, DQ317257.1, L28146.1, AB239939.1, AB188507.1, AB188506.1, AB119643.1, and AB119642.1; CVA4, GQ253377.1, GQ253375.1, GQ253374.1, AB268278.1, AB268278.1, AF081295.1, AB162723.1, AB114087.1, AB234330.1, AB188508.1, AB167797.1, AB239940.1, EU908146.1, EU9081391, EU908133.1, EU908121.1, DQ317264.1, AF290899.1, AF252189.1, GQ176232.1, GQ176231.1, and AB457644.1; CVA5, GQ253378.1, DQ317241.1, DQ317243.1, DQ317245.1, DQ317247.1, DQ251347.1, and AF081296.1; CVA10, AB109018.1, AB119638.1, AB119639.1, AB126614.1, AB162727.1, AB162728.1, AB162730.1, AB167985.1, AF081296.1, AF081300.1, AY694120.1, AY956574.1, AY956577.1, DQ317288.1, DQ317289.1, EU077514.1, GQ214172.1, GQ214173.1, GQ214176.1, GQ214177.1, GU248506.1, GU248509.1, GU248522.1, GU248522.1, and GU248522.1.