Complex genetic variation has been known to occur during the transmission of human enterovirus (HEV), and the HEV virulence and pathogenicity enhanced by genetic recombination also pose a serious threat to human health. In recent years, the interest in recombination mechanism of genetic plasticity has been renewed with the emergence of pathogenic recombinant circulating vaccine-derived polioviruses, which were implicated in poliomyelitis outbreaks in several regions of the world with insufficient vaccination coverage. This paper reviews recent research progress in HEV genome, including evolutionary characteristics, recombination types, and in vitro recombinant construction.
Animals ; Biomedical Research ; trends ; Enterovirus ; classification ; genetics ; Enterovirus Infections ; virology ; Humans ; Recombination, Genetic ; Viral Proteins ; genetics

Animals ; Enterovirus A, Human ; genetics ; Enterovirus Infections ; virology ; Humans ; Reverse Genetics ; methods

OBJECTIVETo study the molecular characterization of CVA9 virus in Tibet.

METHODSTo isolate the enteroviruses from stool specimens of AFP cases and other children in Tibet in 1999-2002, and identify them by neutralization test using the RIVM antiserum; then determine the complete nucleotide sequence of VP1 region of CVA9 viruses, and analyze the results.

RESULTSA total of 10 strains of CVA9 virus were isolated from the stool specimens and identified. The complete nucleotide sequence of VP1 region of these CVA9 viruses were 906nt coding 302 amino acids. To compare with the sequences of the 10 strains of Tibet, the homogeneity of nucleotide sequence were 79.0% - 99.9%; while they were 75.7% - 78.7% compared with Griggs. The phylogenetic tree of CVA9 viruses showed 2 groups, and the isolates from Tibet belong to 1, 2 groups.

CONCLUSIONThe deduction is that the 10 strains are proposed 2 different groups, the strains epidemic in 1999 belong to group 2,while strains in 2000 belong to group 1.

Capsid Proteins ; genetics ; Enterovirus ; classification ; genetics ; Humans ; Phylogeny ; Tibet

China ; Enterovirus ; classification ; genetics ; Phylogeny ; RNA, Viral ; genetics

OBJECTIVEThis report presented an overview on the epidemiology of enterovirus in Yunnan province, the People's Republic of China.

METHODSA total of 210 strains of non-polioviruses isolated under acute flaccid paralysis surveillance during a 5-year study period from 1997 to 2000 and 2004 were examined. Of the 210 non-polioviruses strains, a total of 12 strains of adenoviruses were serologically identified. The remaining 198 isolates were used for molecular typing, and the viral genomes of 195 nonpolio enteroviruses (NPEVs) were translated to corresponding amino acid sequences and compared with those of the prototype strains.

RESULTSBased on molecular typing, 5 isolates were classified into 5 serotypes of human enterovirus A species while 158 isolates into 34 serotypes of B and 32 isolates into 6 serotypes of C species. However, we did not isolate any viruses which belonged to human enterovirus D species. Thus, under acute flaccid paralysis surveillance, human enterovirus B species accounted for 75.2% of the 210 isolates and was considered as the predominant one, followed by human enterovirus C (12.2%), adenovirus (5.7%), and human enterovirus A (2.4%).

CONCLUSIONAlthough the epidemiological characteristics of NPEVs from Yunnan province remained "unknown", the molecular typing method had provided us a breakthrough to understand the epidemiology of these viruses.

China ; epidemiology ; Enterovirus ; classification ; genetics ; isolation & purification ; Enterovirus Infections ; epidemiology ; Genes, Viral ; Humans ; Molecular Epidemiology ; Serotyping

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

To study the effect of miR-490 on Coxsackievirus B3 (CVB3) replication, HeLa cells were trans- fected with miR-490 in vitro, and infected with a Renilla luciferase (RLuc)-expressing CVB3 variant (RLuc-CVB3). The activities of RLuc in these cells were measured at 8h intervals from 0 to 40 h post-infection (p.i.), and the effects of miR-490 on RLuc-CVB3 replication were observed. In a further study, HeLa cells were transfected with either miR-490 or antisense miR-490 (AMO-miR-490), and were then infected with an enhanced green fluorescence protein (EGFP)-expressing CVB3 variant (EGFP-CVB3). The replication of EGFP-CVB3 was then determined by detecting the expression of EGFP. We observed that miR-490 could significantly inhibit the expression of RLuc in infected cells at 32 h p. i. Furthermore, in HeLa cells infected with EGFP-CVB3 at 32 h p.i., EGFP expression was also significantly inhibited by the presence of mniR-490. The inhibitory effect of miR-490 on EGFP expression in EGFP-CVB3-infected cells could be reversed by tranfection with AMQ-miR-490. These results indicated that miR-490 significantly inhibits the replication and expression of QVB3.
Enterovirus B, Human ; genetics ; physiology ; Enterovirus Infections ; genetics ; metabolism ; virology ; HeLa Cells ; Humans ; MicroRNAs ; genetics ; metabolism ; Virus Replication

Objective: To develop RT-nPCR assays for amplifying partial and complete VP1 genes of human enteroviruses (HEVs) from clinical samples and to contribute to etiological surveillance of HEV-related diseases. Methods: A panel of RT-nPCR assays, consisting of published combined primer pairs for VP1 genes of HEV A-C and in-house designed primers for HEV-D, was established in this study. The sensitivity of each RT-nPCR assay was evaluated with serially diluted virus stocks of five serotypes expressed as CCID Results: The sensitivity of RT-nPCR assays for amplifying partial VP1 gene of HEVs was 0.1 CCID Conclusion This RT-nPCR system is capable of amplifying the partial and complete VP1 gene of HEV A-D, providing rapid, sensitive, and reliable options for molecular typing and molecular epidemiology of HEVs in clinical specimens.
Capsid Proteins/genetics* ; Enterovirus A, Human/genetics* ; Enterovirus B, Human/genetics* ; Enterovirus C, Human/genetics* ; Enterovirus D, Human/genetics* ; Humans ; Molecular Epidemiology/methods* ; Molecular Typing/methods* ; Reverse Transcriptase Polymerase Chain Reaction/methods*

OBJECTIVEKnowledge of an enterovirus genome sequence is very important in epidemiological investigation to identify transmission patterns and ascertain the extent of an outbreak. The MinION sequencer is increasingly used to sequence various viral pathogens in many clinical situations because of its long reads, portability, real-time accessibility of sequenced data, and very low initial costs. However, information is lacking on MinION sequencing of enterovirus genomes.

METHODSIn this proof-of-concept study using Enterovirus 71 (EV71) and Coxsackievirus A16 (CA16) strains as examples, we established an amplicon-based whole genome sequencing method using MinION. We explored the accuracy, minimum sequencing time, discrimination and high-throughput sequencing ability of MinION, and compared its performance with Sanger sequencing.

RESULTSWithin the first minute (min) of sequencing, the accuracy of MinION was 98.5% for the single EV71 strain and 94.12%-97.33% for 10 genetically-related CA16 strains. In as little as 14 min, 99% identity was reached for the single EV71 strain, and in 17 min (on average), 99% identity was achieved for 10 CA16 strains in a single run.

CONCLUSIONMinION is suitable for whole genome sequencing of enteroviruses with sufficient accuracy and fine discrimination and has the potential as a fast, reliable and convenient method for routine use.

Child, Preschool ; Enterovirus ; genetics ; Enterovirus A, Human ; genetics ; Enterovirus Infections ; virology ; Feces ; Genome, Viral ; Hand, Foot and Mouth Disease ; virology ; Humans ; Nucleic Acid Amplification Techniques ; instrumentation ; methods

We wished to understand the genetic recombination and phylogenetic characteristics of human en- terovirus A71 (EV-A71) and to explore its potential virulence-related sites. Full-length genomes of three EV-A71 strains isolated from patients in Chenzhou City (China) were sequenced and analyzed. Possible re- combination events and crossover sites were analyzed with Recombination Detection Program v4. 1. 6 by comparison with the complete genome sequences of 231 strains of EV-A71. Similarly, plot and bootscanning analyses were undertaken with SimPlot v3. 5. 1. Phylogenetic trees based on the sequences of VP1 regions were constructed with MEGA v5. 2 using the Kimura two-parameter model and neighbor-joining method. Results suggested that recombination events were detected among the three EV-A71 isolates from Chenzhou City. The common main parent sequence was from JF799986 isolated from samples in Guang- zhou City (China) in 2009, and the minor parent sequence was TW/70516/08. Intertypic recombination e- vents were found in the C4b strain (strain SHZH98 isolated in 1998) and C4a strain (Fuyang strain isola- ted in 2008) with the prototype strains of CVA4 and CVA14 in the 3D region. The chi-square test was used to screen-out potential virulence-related sites with nucleotide substitutions of different types of hand, foot, and mouth disease (HFMD) cases using SPSS v19.0. Results suggested that there were no significant nucleotide substitutions between death cases and severe-HFMD cases. Eighteen significant nucleotide substitutions were found between death/severe-HFMD cases and mild-HFMD cases, and all these 18 substitutions were distributed only in P2 and P3 regions. Intertypic recombination among the predominant circulating EV-A71 strains in the Chinese mainland and other EV-A strains probably dates before 1998, and intratypic recombination might have occurred frequently in the HFMD outbreak from 2008 to 2012. Substitutions in the non-capsid region may be correlated with the changes in virulence of EV-A71. These data suggest that researchers should pay more attention to the relationships between substitutions in the noncapsid region and the virulence of the virus.
Enterovirus A, Human ; genetics ; pathogenicity ; Mutation ; Phylogeny ; Recombination, Genetic ; Virulence