<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

This study aimed to analyse the genetically characterize isolates of Echovirus 11 from Longyan City,Fujian Province,and to reveal their genetic relationships with other isolates from China and abroad. Cerebrospinal fluid specimens from patients diagnosed with viral encephalitis or central nervous system (CNS) infections were collected from Longyan First Hospital between January and December 2011. Seven Echo11 strains were isolated and identified using the RIMV serum panel. The entire VP1 coding regions of four strains were sequenced and typed as Echo11 by an online blast program and,subsequently, phylogenet- ic analyses of the VP1 sequences of these stains and others published on GenBank were conducted. There were 600 nucleotides (nt) in each complete VP1 coding region that encoded 200 amino acids (aa). Among those four Echo11 strains, the sequence identities of nt and aa were 100% and 99%-100% respectively. And phylogenetic analyses indicate belong to subtype DS, the homology compared with DS strain (GU393713) were 93% (nt) and 99% (aa). The sequence identities for the nt and aa were 75%-76% and 90%, respectively, between the current isolates from Longyan and the Gregory prototype strain found in 1953. The sequence identity of nt and aa between the Longyan virus strains and the domestic Shandong strains isolated in 2010 were lower, at 74% and 88%-89%, respectively. However,the highest level of ho- mology was found when the Longyan strains were compared with the Netherlands strain (GU393773) found in 2007 (nt and aa identity: 94%-95% and 98%-99%, respectively). The relatively low levels of similarity between domestic isolates suggest that different transmission routes exist for Echo11 in mainland China.
Adolescent ; Amino Acid Sequence ; Base Sequence ; Child ; Child, Preschool ; Chin ; China ; Encephalitis, Viral ; virology ; Enterovirus B, Human ; chemistry ; classification ; genetics ; isolation & purification ; Enterovirus Infections ; virology ; Female ; Genotype ; Humans ; Male ; Middle Aged ; Molecular Sequence Data ; Phylogeny ; Sequence Alignment ; Viral Proteins ; chemistry ; 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

PURPOSE: This study investigated the possible relationship between viral infection and first trimester pregnancy loss. MATERIALS AND METHODS: A prospective study was performed on 51 gravidas with missed abortion, fetal anomaly, pre-term delivery, and full-tem delivery at Hanyang University Hospital. Enteroviruses were detected by semi-nested reverse transcription-polymerase chain reaction (RT-PCR) and immunohistochemistry in abortive tissues and placentas. Enterovirus serotypes were confirmed by genome sequencing. Herpesviruses were detected by PCR. RESULTS: Coxsackievirus B3 (CVB3) was detected in 8 of 14 missed abortion cases, 1 of 27 full-term cases, and none of the 9 pre-term cases. Coxsackievirus B1 (CVB1) was detected in an encephalocele case. Herpes simplex virus type 1 was found in 4 full-term cases, 3 pre-term cases, and none of the missed abortion cases. CONCLUSION: The prevalence of CVB3 was significantly higher in missed abortion cases compared to full-term or pre-term delivery cases. CVB infection may therefore be an important etiological agent of missed abortion.
Abortion, Missed/*etiology ; Adult ; Coxsackievirus Infections/complications/*diagnosis/virology ; Enterovirus B, Human/genetics/*isolation & purification ; Female ; Humans ; Immunohistochemistry ; Placenta/virology ; Pregnancy ; Pregnancy Complications, Infectious/*virology ; Pregnancy Trimester, First ; Prevalence ; Prospective Studies ; Republic of Korea ; Reverse Transcriptase Polymerase Chain Reaction ; Sequence Analysis, DNA ; Uterus/*virology

Human Enterovirus HEV 74 is a new member of species Human enterovirus B (HEV-B). To understand its evolution and restructuring characteristics, we report the complete genome sequence of a HEV74 strain 05293/SD/CHN/2005(abbreviated as 05293) isolated from an acute flaccid paralysis (AFP) case in Shangdong Province, China, 2005. Analysis of the complete genomic sequence of 05293 showed that its genome was collinear with that of previously described 2 HEV74 strains, except for insertions and deletions at the 5'NTR and the 3 NTR regions. The complete genome sequence of strain 05293 displayed 80. 8% nucleotide and 96% amino acid identity to the prototype strain USA/CA75-10213, and 80. 6% and 95. 9% to another isolated strain Rikaze-136. The P1, P2 and P3 coding regions of strain 05293 displayed 81. 5%, 80. 0%, 79. 7% nucleotide and 95. 9%, 96. 0%, 96.2% amino acid identity to the prototype strain USA/CA75-10213, and 81. 9%, 78. 8%, 79. 5% and 95. 9%, 96. 1%, 95. 7% to strain Rikaze-136, respectively. The phylogenetic tree and Simplot analysis on 05293 and HEV-B genome sequences were performed, and the result indicated frequent recombination within HEV-B.
3' Untranslated Regions ; genetics ; 5' Untranslated Regions ; genetics ; Base Sequence ; China ; Enterovirus B, Human ; classification ; genetics ; isolation & purification ; Enterovirus Infections ; virology ; Evolution, Molecular ; Genome, Viral ; genetics ; Humans ; Muscle Hypotonia ; Paralysis ; virology ; Phylogeny ; RNA, Viral ; genetics ; Recombination, Genetic ; Sequence Alignment ; Sequence Analysis, DNA

Acute myopericarditis is usually caused by viral infections, and the most common cause of viral myopericarditis is coxsackieviruses. Diagnosis of myopericarditis is made based on clinical manifestations of myocardial (such as myocardial dysfunction and elevated serum cardiac enzyme levels) and pericardial (such as inflammatory pericardial effusion) involvement. Although endomyocardial biopsy is the gold standard for the confirmation of viral infection, serologic tests can be helpful. Conservative management is the mainstay of treatment in acute myopericarditis. We report here a case of a 24-year-old man with acute myopericarditis who presented with transient effusive-constrictive pericarditis. Echocardiography showed transient pericardial effusion with constrictive physiology and global regional wall motion abnormalities of the left ventricle. The patient also had an elevated serum troponin I level. A computed tomogram of the chest showed pericardial and pleural effusion, which resolved after 2 weeks of supportive treatment. Serologic testing revealed coxsackievirus A4 and B3 coinfection. The patient received conservative medical treatment, including nonsteroidal anti-inflammatory drugs, and he recovered completely with no complications.
Acute Disease ; *Coinfection ; Coxsackievirus Infections/complications/diagnosis/therapy/*virology ; Echocardiography, Doppler ; Electrocardiography ; Enterovirus A, Human/*isolation & purification ; Enterovirus B, Human/*isolation & purification ; Humans ; Male ; Myocarditis/diagnosis/therapy/*virology ; Pericardial Effusion/diagnosis/therapy/*virology ; Pericarditis, Constrictive/diagnosis/therapy/*virology ; Pleural Effusion/diagnosis/therapy/*virology ; Tomography, X-Ray Computed ; Treatment Outcome ; Young Adult

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

OBJECTIVE: To study the mechanism of osteopontin (OPN) in viral myocarditis by observing the expression of OPN and collagen I (Col I) in mice myocardium. METHODS: The viral myocarditis models were achieved by infection with myocarditic coxsackievirus B3 (CVB3). The myocardium of mice was stained by HE and Masson staining, and the pathological scores and the collagen volume fraction (CVF )of myocardium were tabulated. The expression of Col I mRNA was measured by RT-PCR. The expression of OPN was detected by RT-PCR and ELISA. RESULTS: The histopathological examination revealed a prevalence of myocardial cell necrosis and obvious inflammation changes at the 7th day post-infection. Subsequently the inflammatory lesions were gradually absorbed. At the 28th day, the inflammatory cells had almost disappeared and obvious fibrosis occurred. The pathological scores and the expression of OPN mRNA were higher than those of the control group (P<0.05), and reached the highest level at the 7th day (P<0.05). From the 14th day, these parameters decreased,reflected also in the ELISA results. At the 7th day and the 14th day, the Col I expression was similar to that of control. Col I expression at the 21th and 28th days was higher than those of the control (P<0.05), and correlated positively to the CVF results. CONCLUSION The OPN mRNA expression increased in acute stage of VMC, and higher than that of the control group when in recovery stage, suggesting that OPN might be related to the inflammatory response in acute stage of, and promote the collagen synthesis of recovery stage.
Animals ; Collagen Type I ; genetics ; metabolism ; Coxsackievirus Infections ; metabolism ; Enterovirus B, Human ; isolation & purification ; Male ; Mice ; Mice, Inbred BALB C ; Myocarditis ; metabolism ; virology ; Osteopontin ; genetics ; metabolism ; RNA, Messenger ; genetics ; metabolism

<b>OBJECTIVEb>To isolate and identify the pathogen that caused an outbreak of viral encephalitis in Henan province in 2010; and to analyze the genetic characteristic of gene viral protein1(VP1) on the viral strains isolated.

<b>METHODSb>During the period of the outbreak of viral encephalitis in Lushan county, Pingdingshan city, Henan province, eight hospitalized patients were recruited in the study. All the patients' feces samples were collected. Three patients' cerebrospinal fluids samples and another four patients' serum samples were collected separately. The virus in the samples were isolated and identified by enterovirus (EV) combined serum. The VP1 gene of the positive isolate was amplified by reverse transcriptase PCR method, and its nucleotide sequence was detected and the genetic evolution was analyzed.

<b>RESULTSb>Fifteen samples were collected in total, including 8 feces samples, 3 cerebrospinal fluids samples and 4 serum samples. The results of Fluorescence Quota PCR detection showed that 11 out of 15 samples were positive; 2 strains of virus were isolated from 2 feces samples and the serotype were all Coxsackie-positive identified by the EV combined serum. The full-length VP1 genetic sequences were all 849 bp, and showed 77.1% - 96.9% similar to the nucleotide and 95.8% - 100% similar to the amino acid of CoxB5. The analysis showed that the genetic evolution tree was just the same with Genotype-D.

<b>CONCLUSIONb>CoxB5 whose genotype was Genotype-D, was the pathogen that caused the outbreak of viral encephalitis in Lushan county, Pingdingshan city, Henan province.

Disease Outbreaks ; Encephalitis, Viral ; virology ; Enterovirus B, Human ; isolation & purification ; Genotype ; Humans ; Reverse Transcriptase Polymerase Chain Reaction

In order to confirm the cause of the outbreak of aseptic meningitis in Zhejiang Province in 2002-2004, trace the pathogen and analyze the molecular characteristics, 271 cerebrospinal fluid (CSF) and faeces specimens were collected from suspected patients. The virus strains from the specimens were isolated with RD and Hep-2 cell lines. The VP1 and VP4/VP2 genes of the isolated viruses were sequenced, and their phylogenetic and homology trees were also constructed. Of the total 271 samples, 78 Echovirus type 30 (E30) strains were isolated. All of the complete VP1 genes in 31 sequenced virus isolates of E30 were composed of 876 nt without any insertion or deletion, encoding 292 amino acids (aa). The identity of nucleotide and amino acid in VP1 gene were 84.7%-86.3% and 92.1%-94.2% between the 31 Zhejiang strains and the prototype strain Bastianni of E30, and 87.1%-99.4% and 96.2%-100% among the 31 Zhejiang strains, respectively. The Zhejiang strains of E30 in the phylogenetic tree of the VP1 gene were attributed into two branches of the G and H genotype, respectively. In G genotype, the Shangdong and Jiangsu E30 strains in 2003 among domestic strains and Ukraine E30 strain in 1999 among overseas strains had maximum similarity with the Zhejiang strains, while H genotype had maximum similarity with the Korea E30 strains in 2008. The phylogenetic tree of the VP4/VP2 genes was similar to that of VP1 gene. The results indicated that the outbreak of aseptic meningitis in Zhejiang Provinec in 2002-2004 was caused by the G and H genotypes of E30 strains existing simultaneously. The H genotype was a new variant strain, which was first isolated in Zhejiang Province in 2002.
Amino Acid Sequence ; Capsid Proteins ; genetics ; Cell Line ; Cerebrospinal Fluid ; virology ; China ; epidemiology ; Disease Outbreaks ; Enterovirus B, Human ; classification ; genetics ; isolation & purification ; Evolution, Molecular ; Feces ; virology ; Genotype ; Humans ; Meningitis, Aseptic ; epidemiology ; virology ; Molecular Sequence Data ; Phylogeny ; Sequence Alignment