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*

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

Viral myocarditis (VMC) is a disease characterized by inflammation of myocardial cells caused by viral infection. Since the pathogenesis mechanism of VMC has not been fully elucidated, the diagnosis and treatment of this disease remains extremely challenging. Non-coding RNAs (ncRNAs) are a class of RNAs that do not encode proteins. An increasing number of studies have shown that ncRNAs are involved in regulating the occurrence and development of VMC, thus providing potential new targets for the treatment and diagnosis of VMC. This review summarizes the possible roles of ncRNAs in the pathogenesis and diagnosis of VMC revealed recently.
Coxsackievirus Infections ; Enterovirus B, Human ; Humans ; Inflammation ; Myocarditis/genetics* ; Virus Diseases/genetics*

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

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

3' Untranslated Regions ; genetics ; 5' Untranslated Regions ; genetics ; Enterovirus B, Human ; genetics ; Genome, Viral ; genetics ; Humans ; Poly A ; genetics

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

<b>OBJECTIVEb>To identify the etiology of an aseptic encephalitis outbreak (ten cases) in a hospital of Xiamen city from 11 to 17 May, 2011.

<b>METHODSb>A total of ten patients' throat swabs, anal swabs and cerebrospinal fluid were collected and detected by RT-PCR for pan-enterovirus. The samples containing detectable pan-enterovirus were tested by PCR with genotype-specific general primers located in VP1 region of enterovirus genotype A, B and C (HEV-A, B and C). The PCR products of VP1 segment were purified and sequenced, and phylogenetic analysis was performed. Meanwhile, the pathogens in those samples were isolated in Vero cell culture. Homologous analysis of VP1 sequences were carried out for the cultured virus samples and the original clinical samples to identify the outbreak etiology.

<b>RESULTSb>Among the ten cases, seven cases were positive for pan-enterovirus nucleic acid. When tested by genotype-specific PCR, the throat and anal swab samples from those 7 patients were positive with HEV-B VP1 primers. Meanwhile, the HEV-B VP1 segments were sequenced and phylogenetic analyzed, which indicated the seven cases were all infected by enterovirus Echo 30. The sequences from those samples had homology of 95.3% - 97.1% with the epidemic strains in Zhejiang, 2004. Out of the seven cases, the sequences of XM2, XM3, XM4, XM8 throat swab samples and XM3, XM6 throat samples showed 99.4% - 100.0% homology which were different from the sequence of XM1, and the homology was 92.8% - 93.4%. Furthermore, the viruses were isolated using Vero cells from XM1, XM2, XM3, XM4 and XM8 throat swab samples, and the VP1 sequence showed more than 99.9% homology with the original specimens.

<b>CONCLUSIONb>The local outbreak of aseptic encephalitis was caused by Echo 30 of enterovirus genotype B, and the epidemic strains may have different genetic background.

Child, Preschool ; China ; epidemiology ; Cross Infection ; epidemiology ; virology ; Disease Outbreaks ; Encephalitis ; epidemiology ; virology ; Enterovirus ; genetics ; Enterovirus B, Human ; genetics ; Female ; Genotype ; Humans ; Male ; Molecular Sequence Data

<b>OBJECTIVEb>To study the variation and evolution rules and analyze the genetic characterization of echovirus 30 during an outbreak of aseptic meningitis.

<b>METHODSb>For all the echovirus 30 isolates during the outbreaks in Zhangqiu city in 2003, complete VP1 genes were determined. Phylogenetic tree reconstruction and pairwise sequence were determined to compare the homological relationship and evolution rules between Zhangqiu strains and the other reported echovirus 30 in GenBank.

<b>RESULTSb>Pairwise sequence comparisoms in VP1 region indicated that there were 98.9%-99.5% identity of nucleotide acid among Zhangqiu strains, and having the highest homology with Taian strains (98.5%-99.0%) isolated in Shandong province. At the same time, when comparing with Zhejiang and Jiangsu strains, the nucleotide homology showed 98.2%-98.9%, relatively low identity with Taiwan strains (87.4%-87.6%) and French strains (89.1%-89.7%) but only 82.4%-82.8% identity when comparing with echovirus 30 prototype strain Bastianni. Data from phylogenetic tree analyses indicated that all the echovirus 30 correlated with aseptic meningitis in China recently came from the same evolution linkage and formed a monophyletic cluster.

<b>CONCLUSIONb>In 2003, there was an circulation of echovirus 30 in Shandong province which causing an endemic of aseptic meningitis in local areas. Data from phylogenetic tree analyses indicated that the antigenic characteristics of echovirus 30 in mainland China might be different from strains early isolated in other continents and formed a new genotype.

China ; epidemiology ; DNA, Viral ; analysis ; Disease Outbreaks ; Enterovirus B, Human ; classification ; genetics ; Enterovirus Infections ; epidemiology ; Genotype ; Humans ; Meningitis, Aseptic ; Phylogeny ; Sequence Analysis, DNA

<b>OBJECTIVEb>To develop a method for detection of coxsackie B virus type 1-6 by RT-PCR.

<b>METHODSb>A pair of primers were designed to amplify all types of coxsackie B virus 1-6 efficiently. The PCR product was hybridized in micro-wells in which 6 type specific oligonucleotide probes had been coated respectively, colorimetric detection was performed to discriminate the types of coxsackie B virus.

<b>RESULTSb>This method was shown to be concordant with the IgM ELISA, 71.7% of anti-coxsackie B positive cases could be detected by RT-PCR.

<b>CONCLUSIONb>The RT-PCR method can type coxsackie B virus efficiently and provides a tool for clinical diagnosis and epidemiological investigation.

DNA Primers ; Enterovirus B, Human ; classification ; genetics ; isolation & purification ; Enterovirus Infections ; diagnosis ; virology ; Enzyme-Linked Immunosorbent Assay ; Humans ; Immunoglobulin M ; blood ; Reverse Transcriptase Polymerase Chain Reaction ; methods