China ; epidemiology ; Enterovirus A, Human ; genetics ; isolation & purification ; Enterovirus Infections ; diagnosis ; drug therapy ; epidemiology ; etiology ; Humans

We analyzed the genetic characteristics of coxsackievirus A4 (CV-A4) based on the entire VP1 coding region. Samples were isolated from patients with acute flaccid paralysis (AFP) in Shaanxi, China from 2006 to 2010. We wished to ascertain the predominant genotype and the relationship between CV-A4 infection and AFP. Sixty-eight non-polio enteroviruses were inoculated onto RD cells (to increase the virus titer) and molecular typing was undertaken. The entire VP1 coding region was amplified. Percentage of CV-A4 was 10.3% (7/68). Analyses of genetic identify and creation of phylogenetic trees revealed that CV-A4 could be classified into A, B and C genotypes. Seven CV-A4 strains from Shaanxi and other CV-A4 strains from China formed an independent evolution lineage located in group 4 and belonged to the C2 sub-genotype. These data suggested that CV-A4 strains of sub-genotype C2 were the predominant genotypes in China. These strains co-evolved and co-circulated with those from other provinces in China, so continued monitoring of CV-A4 (by clinical and genetic surveillance) should be enhanced.
China ; Enterovirus A, Human ; classification ; genetics ; isolation & purification ; Enterovirus Infections ; virology ; Genotype ; Humans ; Paralysis ; virology ; Phylogeny ; Viral Proteins ; genetics

To study on the phylogenetic characterization of the VP1 genes of coxsackievirus A16 (CVA16) causing hand-food-mouth disease (HFMD) isolated from Anhui province in 2014. A total of 413 throat swab specimens from HFMD patients were collected during January to November, 2014 for the isolation and identification of enteroviruses using real-time RT-PCR assays. The VP1 regions of CVA16 isolates were amplified using RT-PCR and sequenced. And the phylogenetic tree was constructed among the VP1 regions of those isolates, the different genotypes and sub-genotypes of CVA16 strains. A total of 97 enteroviruses were isolated from 413 samples, the positive rate was 23.49% (97/413), including seventeen CVA16, seventy six HEV71 and four other enteroviruses. The results of the phylogenetic tree showed that 17.CVA16 strains isolated from Anhui in 2014 clustered within B1b evolution branch of B1 genotype. The nucleotide and amino acid sequence identities were 95.30%-100% and 98.70%-100% among the isolates, respectively, but within B1b branch of 17 strains formed several small transmission chains. The nucleotide acid of 17 CVA16 isolates in Anhui province were closed to the strains isolated from Yunnan, Hunan, Guangdong, Tibet and Jiangsu, especially from Hunan in 2013 and from Shenzhen of Guangdong in 2014, the identity were 96.40%-99.70%. The CVA16 strains isolated from Anhui in 2014 were all belong to genetic subtype B1b of B1 genotype was dominant, and among those isolates, several small virus transmission chains had formed with co-circulating and evolution.
China ; Enterovirus A, Human ; classification ; genetics ; isolation & purification ; Enterovirus Infections ; virology ; Genotype ; Humans ; Molecular Sequence Data ; Phylogeny ; Viral Proteins ; genetics ; metabolism

This research firstly reported the molecular analysis of ECHO25 (Entric Cytopathic Human Orphanviruses Type 25). To clarify molecular characteristics of the ECHO25 virus isolates in Henan Province and its relationship with the rest of world's isolates,the complete VP1 sequences of the 4 isolates in Henan were successfully amplified by RT-PCR and were compared with other ECHO25 isolates available from GenBank. Compared with the prototype strain JV-4, the nucleotide sequence identity was 79.2%-80.1%, and the amino sequence identity was 89.0%-92.4, the nucleotide sequence identity among the 4 strains isolates in Henan Province was 93.0%-99.0%, the amino sequence identity was 92.4%-97.5%. HN-01 and HN-26 strains had the highest level of homology, the nucleotide homology was 99.0%; All the 4 strains belonged to the B1 genotype.
China ; epidemiology ; Enterovirus B, Human ; classification ; genetics ; isolation & purification ; Enterovirus Infections ; epidemiology ; virology ; Humans ; Molecular Sequence Data ; Phylogeny

This report presents an overview of human enterovirus B species in Henan Province. A total of 14 isolates of HEV-B species isolated under HFMD surveillance network during the six months in 2010 were examined. Based on molecular typing results targeting VP1 region, 14 isolates were classified into 6 serotypes of HEV-B. Furthermore, comparison of these 14 isolates with reference strains and strains in mainland China was conducted. The phylogenetic analysis revealed that E25, E11 and E6 showed homology with those from Shandong Province which adjoins Henan Province. E1 and E13 showed homology with those from Yunnan Province, and E30 showed homology with Henan strain isolated in 2008. Cocirculation of two lineages of echovirus 6 was observed.
China ; epidemiology ; Enterovirus B, Human ; classification ; genetics ; isolation & purification ; Enterovirus Infections ; epidemiology ; virology ; Feces ; virology ; Humans ; Molecular Sequence Data ; Phylogeny

Echovirus 6, Human ; genetics ; isolation & purification ; Enterovirus Infections ; virology ; Genes, Viral ; Humans ; Sequence Analysis

We wished to analyze the genetic characterization of echovirus 11 (Echo11) from samples of environmental sewage in Shandong Province (China). The VP1 coding region was typed as the strains were amplified. Phylogenetic analyses on the VP1 sequences from these isolates, strains isolated from AFP cases in the period 1994-2010 and others published in GenBank were conducted. From 2011 to 2012, 94 Echo11 strains were isolated from samples of environmental sewage in Jinan and Linyi City in Shandong Province. Numbers of Echo11 were seasonal and reached peaks in the summer and autumn in both cities; A- mong these isolates, nucleotide (nt) identities were 89.5%-100.0% whereas amino acid (aa) identities were 95.4%-100.0%. The nt and aa identities were 76.6%-79.7% and 90.4%-92.5% between those strains and the prototype (Gregory) strain of Echo11, respectively. All isolates from Shandong Province were the A genotype and the strains evolved very rapidly, which suggested that several transmission chains was co-circulating. We described the temporal fluctuation and genetic characterization of Echo11 isolates from surveillance of environmental sewage in Shandong Province, thereby providing important information for exploring the dynamic change and genetic variation of circulating human enteroviruses in this Province in China.
China ; Enterovirus B, Human ; classification ; genetics ; isolation & purification ; Phylogeny ; Sewage ; virology

Based on RSCU method and by analyzing the preference of codon usage in VP1 nucleotide sequences of EV71 isolated in Chinese mainland and Taiwan region from 1998 to 2008, it is clear that there is an obvious time discrimination in RSCU calculated from EV71 VP1 strain between two different regions of China and it is more obvious in Taiwan region, therefore, according to the diversity of RSCU, the years can be divided into 2 intervals in Chinese mainland and 4 intervals in Taiwan region, especially, the number of intervals in one region have a positive co-relation with the activity of variation of the EV71 in the same region. The change of the preference of codon usage in VP1 nucleotide sequences of EV71 can significantly embody the Variation of the EV71, so we can make use of the analysis on preference of codon usage in VP1 nucleotide sequences of EV71 to predict the possible variation trend of the EV71.
Base Sequence ; China ; Codon ; Enterovirus A, Human ; chemistry ; classification ; genetics ; isolation & purification ; Enterovirus Infections ; virology ; Genetic Variation ; Humans ; Molecular Sequence Data ; Taiwan ; Viral Proteins ; chemistry ; genetics

Genetic characteristics of enterovirus type 71 in Shenzhen from 2005 to 2008 were analyzed. All samples were detected by RT-PCR using EV71-specific primers. The VP1s of EV71 strains were amplified and sequenced. A phylogenetic tree was constructed by comparison of the sequences with those of subgenotype A, B and C using DNASTAR, BioEdit and Mega 3.1 softwares. The VP1 nucleotide sequences of 17 strains of Shenzhen shared 95.3%-99.4% identities with cluster C4a, and one strain shared 93.0%-95.6% identities with cluster C4b. The homogeneity of the VP1 nucleotide sequence with subgenotype A and B was 81.8%-86.0%. Among 18 strains, the homogeneity of the VP1 nucleotide sequence was between 92.5%-100%. All EV71 strains circulating in Shenzhen were of subgenotype C4. The predominant strain of EV71 belonged to cluster C4a, also there was cluster C4b.
Cell Line ; China ; Enterovirus A, Human ; classification ; genetics ; isolation & purification ; Enterovirus Infections ; virology ; Feces ; virology ; Genotype ; Humans ; Molecular Sequence Data ; Phylogeny ; Viral Proteins ; genetics

To construct a recombinant expression plasmid Bacmid-P1-3CD containing the P1 and 3CD genes of enterovirus 71(EV71), the P1 and 3CD genes were cloned into the same baculovirus shuttle vector (Bacmid). Recombinant AcMNPV-P1-3CD was obtained by transfecting the Bacmid-P1-3CD into the insect cell line of S f9. With the IFA and Western-blot methods for identification of expression products confirmed that the target protein was expressed in interior of infected S f9 cells. Electron microscopy showed that the structural protein capsid P1 was cut by virus-encoded protease 3CD and assembled into EV71 virus like particles (VLPs) about 27nm diameter. Different values of MOI and time points of expression were compared to explore the optimal expression condition, and the results showed that the time point could be a more important factor. Then we used S f9 cells with serum-free medium in CellSTACK-10 Culture Chambers to produce EV71 VLPs in the confirmed condition. After purification of VLPs by density gradient centrifugation, we observed on SDS-PAGE profile the purified sample contained three major proteins whose molecular masses corresponded to those of VP1 (39kD), VP0 (34kD) and VP3 (26kD) as well as the intact structure, which can be greatly used for further study in protein structure and genetic engineering vaccine research.
Animals ; Baculoviridae ; genetics ; metabolism ; Cell Line ; Enterovirus A, Human ; genetics ; isolation & purification ; physiology ; ultrastructure ; Gene Expression ; Spodoptera ; Viral Proteins ; genetics ; metabolism ; Virion ; genetics ; isolation & purification ; physiology ; ultrastructure ; Virus Assembly