| Objective:To investigate the prevalence features of different pathogens of hand, foot and mouth disease (HFMD),and to master and clarify the pathogenic spectrum and the genetic characteristics of HFMD in Hebei Province, in order to provide scientific evidence for the prevention and control of HFMD.Methods:1 According to epidemiological investigation, stool specimens, anal swab specimens, throat swab specimens, vesicle fluid specimens and cerebrospinal fluid specimens were collected from clinically diagnosis cases with HFMD.2 The consensus and type-specific primer pairs were designed respectively according to the conservative gene sequences of 5'-UTR, the VP1 and 2A gene sequences. And specimens from cases with HFMD clinically diagnosised were detected by RT-PCR, and the PCR products were identified by agarose gel electrophoresis.3 EV was isolated with RD cells from EV positive specimens in laboratory diagnosis cases with HFMD. The specimens with CPE appearance were detected by RT-PCR with the primer pairs for EV.4 The VP1 of 18 EV71 and 9 CA16 strains were amplificated by RT-PCR with specific primer pairs for VP1 entire gene sequences, then the PCR amplification products were sequenced to perform nucleotide sequence homology analysis and construct the phylogenetic tree with DNASTAR software for analyzing the gene subtype and variation in Hebei province.Results:1 1990 specimens from clinical diagnosis cases with HFMD were detected by RT-PCR.1296 specimens were EV positive(65.13%), of which 752 specimens were EV71(37.79%),452 were CA16(22.71%),1 was EV71+CA16 (0.05%) and 91 were the other enterovirus (4.57%). The results of constituent ratio of different pathogen showed that fristly EV71 was the principal pathogen of the cases, accounting for 58.03% (752/1296)of positive cases, then followed by CA16 and accounted for 34.88%(452/1296) of positive cases, the other EV occupied a certain proportion and accounted for 7.02%(91/1296) of positive cases.55 other EV-positive specimens were selected randomly for further analysis by RT-PCR and sequence.31 samples were identified in addition to 24 specimens non typing.From the view of composition, CA10 accounted for 18.18%(10/55), while CA6 accounted for 14.55%(8/55), ECO30 accounted for 9.09%(5/55), ECO6 and ECO9 accounted for 3.64% respectively, and CA5, CA14, CB5, ECO14 accounted for 1.82% respectively, and about 43.64% was not typed. The results suggested that the pathogen of other EV was complex, involving a variety of viruses (9 species were detected in this experiment), and mainly CA10, CA6, ECO30.2 In the view of different regions of the pathogenic situation, Qinhuangdao, Handan, Baoding, Xingtai had a high proportion of EV71, about 77.53%,76.18%,69.12%,65.98%respectively; while Hengshui, Langfang, Cangzhou had a high proportion of CA16 relatively, about 64.29%,58.70%,51.28% respectively; and Cangzhou had a high proportion of other EV, about 33.33%. By further typing, except for Langfang, Xingtai, Qinhuangdao, there was one or more of the other type in the other regions. Of 11 samples in Cangzhou,6 were CA10 and 1 was ECO30; there were more other types of EV type in Shijiazhuang, and seven kinds of other types were detected out of 13 samples, of which 1 was CA5,1 was CA6,1 was CA10,1 was CA14,2 were ECO6,2 were ECO9 and 3 were ECO30; at the same time four kinds of type were detected out of 7 samples in Handan, and CA6, CA10, ECO30, ECO14 were detected respectively. Pathogenic form showed that the higher CA10 positive separation proportion was in Cangzhou, accounting for 54.55%(7/11), while a higher proportion of ECO30 in Shijiazhuang, which accounted for 23.08%(3/13). But there were still 24 samples unidentified.31 230 mild cases were detected,689 cases were positive(56.02%), of which 362 were CA16,260 were EV71 and 67 were other enterovirus. The rate of CA16, EV71and other enterovirus was 52.46%,37.74%and 9.72%respectively and the ratio of them was 3.9:5.4:1. The 42 EV positive cases from 67 other EV cases were further sub-typed, which showed that CA10 accounted for 19.05%(8/42), CA6 accounted for 16.67%(7/42), ECO30 accounted for 7.14%(3/42), ECO9 ccounted for 4.76%(2/42), CA5, CB5, CA14, ECO14 accounted for 2.88%(1/42) respectively, and about 42.86%(18/42) was not typed.747 severe cases were detected,594 cases were positive(79.52%), of which 1 was EV71+ CA16,479 were EV71,90 were CA16 and 24 were other enterovirus, the rate of them was 0.17%,77.13%,14.36%and 8.35%respectively, the ratio among EV71, CA16 and other enterovirus was 20.0:3.8:1. The 13 EV positive cases from 24 other EV cases were further sub-typed, which showed that ECO6, ECO30, CA10 accounted for 15.38%(2/13) respectively, CA6 accounted for 7.69%(1/13), and about 46.15% (6/13)was not typed.13 death cases were detected, all of them were EV71 positive.4 141 stool specimens were detected by RT-PCR, of which 101(71.63%) were EV positive; 617 anal swab specimens were detected and 514(83.31%) were EV positive; 90 vesicle fluid specimens were detected and 53 (58.89%) were EV positive; 1141 throat swab specimens were detected and 628(55.04%) were EV positive; there was no EV positive detected in cerebrospinal fluid specimens.5 EV was isolated with RD cells from 160 EV positive specimens, 57 strains were EV positive, the separation rate was 35.63%. Identified by RT-PCR and sequence,32 strains were EV71,9 strains were CA16,8 strains were CA16+EV71,4 strains were CA10,1 strain was CA6 and 1 was CA5.17 stool specimens were inoculated, and 9 were isolated with separation rate 52.94%; 42 rectal swab samples were inoculated, and 15 were isolated with separation rate 35.71%; 11 vesicle fluid specimens were inoculated, and 3 were isolated with separation rate 27.27%; 89 throat swab specimens were inoculated, and 30 were isolated with separation rate 33.71%; 1 cerebrospinal fluid specimen was inoculated without EV isolated.6 The homogeneity of VP1 nucleotide sequence of the 18 separated EV71 strains from Hebei Province was 94.4%-99.8%; with isolates from 9 provinces and cities nationwide being 94.5%-99.6%; with the representative isolates of A and B genotypes being 81.7%-82.7%, 83.2%-85.1% respectively; with the representative isolates of C genoty-pes being 86.6%-99.6%; with the representative isolates of C1,C2,C3,C4 and C5 subgenotypes being 89%-90.1%,88.1%-90.2%,88%-89.2%, 91.9%-99.6%,86.6%-88.2% respectively.7 The homogeneity of VP1 nucleotide sequence of the 9 separated CA16 strains from Hebei Province was 91.6%-99.9%; with the representative isolates of A genotypes being 75.4%-77% respectively; with the representative isolates of 33 B genotype representative strains being 88.6%～99%.8 The EV71 strains belonged to C4a evolution branch of C4 subgenotype and CA16 belonged to genotype B by analyzing the EV71 and CA16 genetic characteristics and phylogenetic tree.9 Of 16 EV71 strains, the amino acid combined model was KADSTV in the six subtype related sites, others were KANSTV and KADSIV.10 17 EV71 strains were highly conserved in the main immune epitopes, the amino acid sequence was EIDLPLEGTTNPNGYA. But in the 93th amino acid of 2009-hebei-SJZ-120F, Isoleucine was replaced by Valine.Conclusion:1 EV71 and CA16 are the primary pathogen of HFMD of Hebei Province in 2009. While there are alse a few of CA10, CA5, CA6, CA14, CB5, ECO6, ECO9, ECO14, ECO30 and other types. EV71 is the principal pathogen of severe and dath cases.2 The EV71 strains belongs to C4a evolution branch of C4 subgenotype and the CA16 belongs to genotype B by analyzing the EV71 and CA16 genetic characteristics and phylogenetic tree.3 Amang all the EV71 strains, the major amino acid combined model is KADSTV in the six subtype related sites, there are also KANSTV and KADSIV, which are the supplements for the similar pattern of KADSTI.4 Most EV71 strains are highly conserved in the main immune epitopes, the amino acid sequence is EIDLPLEGTTNPNGYA.5 Of all CA16 strains, there are two significant mutations, which may has an effect on the conformation of the antigen.
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