From 1999 to 2004, there were no epidemics of HFMD reported in Shenzhen, but each year there were small, local outbreaks associated with only a few cases of neurological disease and no reported fatalities. For this study, 147 stool specimens were collected, and the case number each year ranged from 22 to 29. The stool specimens were reverse transcription-PCR screened with EV71- and CA16-specific primers. We identified 60 positive specimens; EV71 was detected in 19 specimens and CA16 in 41 specimens. Coinfection by EV71 and CA16 was not identified in these samples. Of the patients with molecularly confirmed EV71 or CA16 infection, the age ranged from 6 months to 8 years, with 52 of the patients (87%) being less than 5 years old. There were 39 boys and 21 girls, for a male-to-female ratio of 1.9 to 1. Interestingly, no EV71 infection was detected in the samples from 1999 and 2000, whereas the samples from 2001, 2002, 2003, and 2004 showed 3, 2, 3, and 11 cases positive for EV71, respectively. CA16 was detected from samples taken in each year from 1999 to 2004, with a distribution of 7, 2, 8, 13, 2, and 9 cases, respectively. In 2004, out of eight positive specimens collected from a single kindergarten in Shenzhen, five were positive for EV71 infection.
We next sequenced and analyzed the EV71 strain viral genomes from the 5′ untranslated region (5′ UTR) and the VP4 and VP2 to VP1 regions (about 3,200 bp). Pairwise nucleotide and amino acid comparisons of the five individual regions showed that the variability was minor among the EV71 strains detected in Shenzhen over the 4-year period (Table ). The nucleotide identities of the partial 5′ UTR (616 bp) sequences were higher than 93.3%. Nucleotide insertions and deletions were observed in this region compared to comparable sequences from the SHZH98 strain and the prototype, BrCr. The VP4 region showed greater than 92.7% nucleotide identity and 100% amino acid identity across the detected EV71 strains. The VP2 region showed the largest variation in amino acid sequence, with identities ranging from 97.2% to 100%. The VP3 region had the highest nucleotide variation (>89.9% identity) but showed amino acid identities higher than 98.3%, with 10 strains detected from four different years yielding identical amino acid sequences. The nucleotide and amino acid identities of the VP1 region were higher than 92.7% and 98.3%, respectively.
Sequence comparisons of genome regions among EV71 Shenzhen strains
Phylogenetic analysis of these strains was based on the alignment of complete VP1 or VP4 gene sequences. A total of 43 EV71 strains were used for phylogenetic analysis of the VP1 gene (Fig. ), including the 19 EV71 strains identified in this study, 9 EV71 strains previously detected from the Chinese mainland (GenBank), and 15 other EV71 strains (GenBank) included as genotype references. Consistent with the results of previous studies, all 43 strains clustered into three distinct genotypes on the phylogenetic tree. The EV71 strains detected from the Chinese mainland were closely related to each other and grouped into genotype C to form a new genetic lineage (C4) distinct from the previously described C1, C2, and C3 lineages. The VP4 gene was similarly analyzed in 30 EV71 strains, including 13 reference strains (Fig. ). As in the VP1 analysis, the VP4 sequences of 17 EV71 strains detected from Shenzhen also fell into genotype C, clustered in the C4 lineage.
FIG. 1. Phylogenetic analysis based on EV71 VP1 nucleotide sequences (891 bp). Details of the EV71 strains included in the dendrogram are provided in Table S1 in the supplemental material. The marker denotes the percentage of bootstrap frequency of the main branch. (more ...)
FIG. 2. Phylogenetic analysis based on EV71 VP4 nucleotide sequences (207 bp). Details of the EV71 strains included in the dendrogram are provided in Table S1 in the supplemental material. The marker denotes the percentage of bootstrap frequency of the main branch. (more ...)
We performed a similar analysis of the CA16 strains detected from Shenzhen during the 6-year period of this study. We sequenced and analyzed genome regions from the 5′ UTR, VP4, VP2, and VP3 to VP1 (about 3,200 bp) and used pairwise nucleotide and amino acid comparisons to assess the heterogeneity among the strains (Table ). The nucleotide identities of the partial 5′ UTR (617 bp) were higher than 90.2%, with nucleotide insertions and deletions noted in comparison to the sequence of the CA16 prototype, G10. In the VP1 region, the nucleotide identities ranged from 88.3 to 100%, and amino acid identities were higher than 95.6%. In 1999 and 2000, the nucleotide differences seldom resulted in amino acid variations. From 2001 to 2004, the accumulated nucleotide changes resulted in new amino acid variations as nonsynonymous substitutions increased. The nucleotide and amino acid identities between two particular strains isolated in 2003 were 99.5 and 99.3%, respectively, with three out of four nucleotide variations forming nonsynonymous substitutions. This tendency was more obvious in VP1 than in VP2 or VP3. The nucleotide identities in the VP4 region ranged from 85.9 to 100%, and the amino acid sequences of 34 strains isolated from six different years were identical. In contrast, the VP4 regions of four strains (SHZH99-11, 99-48, 99-79, and 00-2) showed higher variation, with identities of >97.1% among the four strains and identities with the other strains ranging from 85.9 to 91.7%.
Sequence comparisons of genome regions among CA16 Shenzhen strains
Phylogenetic analysis of the CA16 strains was based on the alignment of complete VP4 gene sequences among the detected strains and 53 additional previously characterized CA16 strains, allowing us to newly clarify the genetic lineages of CA16 strains detected from 1951 to 2004 in the United States, the United Kingdom, Japan, Malaysia, People's Republic of China, and Taiwan. While there were a few outliers, the CA16 strains clustered in three distinct genetic lineages (A, B, and C) supported by the indicated bootstrap values (Fig. ). Lineage A contains the CA16 prototype, G10, and a single isolate from Japan (G10JPN). The nucleotide identity of the two strains was 99.5%, and the nucleotide sequences differed from all other strains by 20.4 to 27.4%. Lineage B (>93% identity) comprised four strains detected in this work during 1999 and 2000, some early Japanese strains, and some Malaysian strains detected in 2000. Most of the Asian strains detected during the 1990s and the United Kingdom strain Epsom/15290/99 belonged to lineage C and shared nucleotide identities higher than 90.8%. The nucleotide divergence between lineages B and C was 7.0 to 18.6%.
FIG. 3. Phylogenetic analysis based on CA16 VP4 nucleotide sequences (207 bp). Details of the CA16 strains included in the dendrogram are provided in Table S1 in the supplemental material. The marker denotes the percentage of bootstrap frequency of the main branch. (more ...)