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

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*

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

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

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

Animals ; China ; epidemiology ; Enterovirus A, Human ; genetics ; physiology ; Enterovirus Infections ; epidemiology ; metabolism ; virology ; Humans ; Receptors, Virus ; genetics ; metabolism

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

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

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