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

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 investigate the etiology of the outbreak of viral encephalitis in Jinan area in 2003.

<b>METHODSb>Virus-specific nucleic acid fragments were amplified by random PCR and RT-PCR using specific primers to enterovirus. After sequencing, the gene sequence was handled by the program BLAST for homologous analysis and the software Clustal W 1.82 for multiple sequence alignment to identify the etiology and its genotype.

<b>RESULTSb>Five strains were isolated from clinical specimens. A gene fragment for one strain was acquired using random PCR, which was highly homologous to enterovirus. Then, the 5' non-translated region and partial VP1 region were amplified and sequenced. The five isolated strains were all identified as Coxsackievirus B5, and what was more, they were most homologous to the strain isolated during the outbreak of aseptic meningitis and encephalitis in Zhejiang province from 2002 to 2004.

<b>CONCLUSIONb>Coxsackievirus B5 is closely associated with the outbreak of viral encephalitis in Jinan area in 2003. It is an important etiology but other viruses may also played a role which remains to be clarified.

China ; epidemiology ; Disease Outbreaks ; Encephalitis, Viral ; virology ; Enterovirus B, Human ; genetics ; isolation & purification ; Humans ; Polymerase Chain Reaction

<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

<b>OBJECTIVEb>To determine the partial sequence of virus strains causing an aseptic meningitis outbreak in northern part of Jiangsu province in 2003 and to compare them with the same serotype strains isolated in other countries to better understand its genetic characteristics and hereditary trend of development.

<b>METHODSb>Virus RNA was amplified using two sets of specific enteroviral 3' half of VP1 primers 012/011 and 040/011. Polymerase chain reaction (PCR) products were purified and sequenced. BLAST program was then used to perform on nucleotide and amino acid pairwise-alignment with all available sequences in NCBI database. Phylogenetic trees were drawed to compare with other enteroviral sequences using PHYLIP software.

<b>RESULTSb>Under BLAST program, three sequences we submitted to GenBank were identically inferred as echovirus type 30, which had been identified by neutralization test in previous study. Phylogenetic analysis demonstrated that strains isolated from this outbreak were aggregated into a cluster, and the closest relationships with them were those isolated in 1999 and 2000. This phenomenon indicated that Echo30 from this outbreak was different from other strains in different epidemic area.

<b>CONCLUSIONb>3' half of VP1 sequence could be used to quickly identify the serotype of isolated enterovirus. Strains isolated from this outbreak had the similar hereditary developing trend comparing with Echo30 strains isolated from other countries.

Base Sequence ; China ; epidemiology ; DNA, Viral ; genetics ; Disease Outbreaks ; Enterovirus B, Human ; genetics ; isolation & purification ; Female ; Humans ; Male ; Meningitis, Aseptic ; epidemiology ; virology ; Molecular Sequence Data ; Sequence Analysis, DNA

An outbreak of hand foot and mouth disease occurred in Shang dong, China in 2009. Almost 20% of patient's swabs was positive for Coxsackie virus B5 (CVB5) identified by RT-PCR and sequencing. It was suggested that CVB5 may be another important pathogen for HFMD. Fifteen pairs of overlapping primers were designed and the genome sequence was sequenced. The genome of CVB5 was 7 399 nt in length, coding for 2 185aa. The genome displayed 80.6%-85.3% nucleotide sequence identity and 96.1%-96.9% amino acid sequence identity with another three CVB5 respectively. Phylogenetic analysis showed that different segment of genome underwent a distinct evolutionary and selective pressure. Simplot analysis displayed no evident recombination between genome of CVB5 and other HEV B viruses. The complete and characterized genome of CVB5/09 provides further insight into the genetics of CVB5 and other HEV B viruses, aiding in the surveillance and control of HFMD.
Base Sequence ; China ; Coxsackievirus Infections ; virology ; Enterovirus B, Human ; classification ; genetics ; isolation & purification ; Genome, Viral ; Humans ; Molecular Sequence Data ; Phylogeny ; Viral Proteins ; genetics

<b>OBJECTIVEb>To provide a feasible and cost-effective next-generation sequencing (NGS) method for accurate identification of viral pathogens in clinical specimens, because enormous limitations impede the clinical use of common NGS, such as high cost, complicated procedures, tremendous data analysis, and high background noise in clinical samples.

<b>METHODSb>Viruses from cell culture materials or clinical specimens were identified following an improved NGS procedure: reduction of background noise by sample preprocessing, viral enrichment by barcoded oligonucleotide (random hexamer or non-ribosomal hexanucleotide) primer-based amplification, fragmentation-free library construction and sequencing of one-tube mixtures, as well as rapid data analysis using an in-house pipeline.

<b>RESULTSb>NGS data demonstrated that both barcoded primer sets were useful to simultaneously capture multiple viral pathogens in cell culture materials or clinical specimens and verified that hexanucleotide primers captured as many viral sequences as hexamers did. Moreover, direct testing of clinical specimens using this improved hexanucleotide primer-based NGS approach provided further detailed genotypes of enteroviruses causing hand, foot, and mouth disease (HFMD) and identified other potential viruses or differentiated misdiagnosis events.

<b>CONCLUSIONb>The improved barcoded oligonucleotide primer-based NGS approach is simplified, time saving, cost effective, and appropriate for direct identification of viral pathogens in clinical practice.

Clinical Laboratory Techniques ; DNA Barcoding, Taxonomic ; DNA Primers ; Enterovirus ; classification ; genetics ; isolation & purification ; Herpesvirus 4, Human ; genetics ; isolation & purification ; Humans ; Influenza B virus ; genetics ; isolation & purification ; Real-Time Polymerase Chain Reaction ; Sequence Analysis, DNA ; methods ; Sequence Analysis, RNA ; methods

A coxsackievirus B strain was successfully isolated by cells culture from cardiac muscle tissues of a dead Sichuan golden monkey with myocarditis from a zoo of Changchun in China. The isolate was consistent with CVB by morphology, physicochemistry test, animal regression test and RT-PCR. Analysis of VP1 partial gene sequence and detection of mice specific serum IgG showed that the strain isolated was a coxsackievirus B3. It was the first CVB case report in Sichuan golden monkey and the strain isolated was named CVB/SGM-05.
Animals ; Cercopithecus aethiops ; Enterovirus B, Human ; isolation & purification ; Haplorhini ; virology ; Heart ; virology ; Mice ; Reverse Transcriptase Polymerase Chain Reaction ; Vero Cells ; Viral Structural Proteins ; genetics

<b>OBJECTIVEb>To ascertain the pathogen of aseptic encephalitis epidemic in Long-Yan city in Fujian, and to find out the genetic characteristics of the virus.

<b>METHODSb>Rapid detection of enteroviral RNA by reverse transcription polymerasechain reaction (RT-PCR) was directly carried out in cerebrospinal fluid(CSF) to isolate and identify the viruses from CSF at the same time, and to detect the neutralization antibody in two serum specimens collected in acute and convalescence phase. Nucleotides of VP1 region was also analyzed by constructing phylogenetic tree.

<b>RESULTSb>ECHO 19 infection was rapidly diagnosed and sequence analysed by RT-PCR, and then echovirus type 19 from 16 of 30 CSF samples (53.33%) was isolated and detected using RD and Hep-2 cells simultaneity. The titer of ECHO 19 neutralization antibody became positive or increased by 4 times from acute to convalescence phase in 4 of the 5 patients. Phylogenetic analyses of the VP1 genes of these isolates showed that their nucleotides identity were 98.9% -100.0% which were different from those ECHO 19 from GeneBank database by 13.0%-22.4%.

<b>CONCLUSIONb>The etiology of the epidemic of aseptic encephalitis was attributed to ECHO 19. The method of molecular identification not only provided rapid diagnosis of enterovirus infections, but also information about the genetic character of the viruses.

Antibodies, Neutralizing ; analysis ; China ; epidemiology ; Echovirus Infections ; diagnosis ; immunology ; virology ; Encephalitis, Viral ; epidemiology ; virology ; Enterovirus B, Human ; genetics ; isolation & purification ; Humans ; Phylogeny ; Reverse Transcriptase Polymerase Chain Reaction