Identification And Genomic Characteristics Of Epidemic Strains Of Enterovirus 71 Isolated From Chongqing Area In 2009

Background:Hand-Foot-mouth disease (HFMD) is caused by a variety of enterovirus which occurs mainly in children under 5 years old, especialy in children under 3 years old. Thera are more than 20 enterovirus which can cause HFMD including EV71 virus, group A of Coxsackie virus and enterovirus ECHO virus. In recent years, EV71 is concerned as the main pathogen of HFMD in some regions of China because of its complications of nervous system. HFMD has been epidemic in Chongqing area since 2008. The pathogen was detected in 77 children with HFMD in 2009. It showed that 41/77 cases (53.2%) was positive for EV71, 6/77 cases (7.8%) was positive for CA16 and 7/77 cases (9.1%) was positive for EV71 and CA16. It indicated that EV71 is the main pathogen of HFMD in Chongqing area.Objective:To understand the genomic sequence characteristics and the possible origins of enterovirus type 71, EV71 strains was isolated from specimens of patients with HFMD in Chongqing area in 2009 and the genome sequences were compared and analyzed with other EV71 in Genebark to know the characteristics of genome sequence and the possible spread sources of EV71 strians in Chongqing area.Methods: (1) One hundred specimens ( including cerebral spinal fluid, vesicle fluid, stool , throat swab and anal swab) were collected from 47 patients with HFMD during the epidemic season in 2009. The specimens were inoculated into RD cells to isolate EV. The supernatant of cytopathic effect (CPE) were detected by RT-PCR with enterovirus universal primers, genus-specific primers of EV71 and genus-specific primers of Coxsackie A16. The amplified PCR products for EV71 were sequenced and sequence was compared with international standard BrCr strain by the biological software of Bioedit. And the virus particles in infected RD cells by isolated strains of EV71 was observed under electron microscopy.(2) RT-PCR was performed to amplify the whole genome of EV71 strains of Chongqing area in 2009 by four linked fragments. PCR amplification products were purified and sequenced assembling fragment into full genome sequence. Nucleotide sequence and amino acid were analyzed by using of biological information software. Phylogenetic tree was drawn to identify the epidemic strain of genetic variation and the possible spread of the source.Results:(1) The apparent cytopathic effect in cell cultures were observed in 7 samples (stool or anal swab) of 100 clinical specimens of children with HFMD, The results of RT-PCR show that four specimens were positive with entrovirus universal primers only, and three were positive results with EV71-specific primers and entrovirus universal primers, while the negative results were obtained with CA16-specific primers for all the samples. The recovery DNA fragment of the PCR products were cloned into DH5αand the sequencing identification confirmed as EV71 virus. And the virus particle was observed in infected RD cells under scanning electronic microscope that was circular with no envelope virus particles, which accordand with the report of the Chinese Center for Disease Control and Prevention (China CDC) , and confirmed that the EV71virus was isolated successfully.(2) The complete genome of the 3 epidemic strains of enterovirus type 71 isolated in Chongqing was 7409, 7406 and 7404 nucleotides, respectively. The homology of VPl regions among 3 strains were from 99.2% to 99.3%.Sequences alignment demonstrated the isolated strains Chongqing2-09-China and Chongqing3-09-China had high homology with viruses from Fuyang, Anhui (2008) and the isolated strain EV71 Chongqing1-09-China had high homology with viruses of 984 polyprotein and 1235 polyprotein in Taiwan (2005). Compared to AF302996 (SHZH98), the homology of VP1 region was more than 91% for each of nucleotide sequence of EV71 of chongqing area , which indicates that the three isolated strains belong to C4 subgenotype. Phylogenetic analysis also showed that the epidemic strains of EV71 in Chongqing area belong to subgenogroup C4.Conclusions:(1) The epidemic strains of EV71 in Chongqing in 2009 was isolated successfully.(2) The epidemic strains of EV71 in Chongqing area in 2009 belong to subgenogroup C4.