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*

OBJECTIVETo study the characteristics of EV71 JN200804 strain infection in one-day old BALBI c mjce and to establish a animal model of EV71 infection , and to provide information and technical support for the evaluation of the EV71 vaccine and antiviral medicine.

METHODSOne-day old BALBic mice were infected with EV71 JN200804 strain through oral( PO) ,intracranial(IC) ,intraperitoneal (IP), intramuscular (IM) routes, respectively. All mice were sacrificed at paralysis of hind limbs and collected organs for viral isolation, RT -PCR and pathological examination, and the electrophysiology were detected before sacrifice.

RESULTSAll mice infected through IC, IP and IM routes were paralyzed in hind limbs at 4-5 days and died at 7 days about, the hypokinesia and lethargy of mice were observed through PO routes. The viruses could be isolated and detected in the muscle from mice infected through IC, IP and IM routes and in the spinal cord through IC routes by viral isolation and RT-PCR. The neurogenic and myogenic disorders were detected by electromyography. Histopathologically, the varied pathologic changes were observed in the mouse cerebellum , spinal cord , muscle , heart, lung, liver and kidney.

CONCLUSIONEV71 JN200804 strain can infect one-day old BALBI c mice and induce paralysis of hind limbs, its animal infection model may apply to study of EV71 infection pathogenesis and antiviral medicine, and evaluation of the EV71 vaccine.

Animals ; Disease Models, Animal ; Enterovirus A, Human ; immunology ; physiology ; Enterovirus Infections ; immunology ; virology ; Mice ; Mice, Inbred BALB C

We have submitted and published the above paper in Infection and Chemotherapy in 2003. Another paper with a condensed but almost same content as the above paper was submitted to and published in an English journal (Acute Hemorrhagic Conjunctivitis Caused by Coxsackievirus A24 Variant, South Korea, 2002. Emerg Infect Dis 2003). Our original intent was to introduce the study to all readers because the two journals seemed to cover different spectrum of readers. However, we did not follow the necessary steps for secondary publication. So we are asking the permission of the Editor to retract the above paper. We hereby regret to have to retract the paper.
Conjunctivitis ; Conjunctivitis, Acute Hemorrhagic* ; Drug Therapy ; Enterovirus C, Human* ; Korea* ; Publications

We have submitted and published the above paper in Infection and Chemotherapy in 2003. Another paper with a condensed but almost same content as the above paper was submitted to and published in an English journal (Acute Hemorrhagic Conjunctivitis Caused by Coxsackievirus A24 Variant, South Korea, 2002. Emerg Infect Dis 2003). Our original intent was to introduce the study to all readers because the two journals seemed to cover different spectrum of readers. However, we did not follow the necessary steps for secondary publication. So we are asking the permission of the Editor to retract the above paper. We hereby regret to have to retract the paper.
Conjunctivitis ; Conjunctivitis, Acute Hemorrhagic* ; Drug Therapy ; Enterovirus C, Human* ; Korea* ; Publications

Animal model is very important for anti-EV71 (enterovirus 71) drug and vaccine development. 1-day-old suckling EV71 mouse model is the main in vivo model used in China. 1-day-old suckling EV71 mouse is too small to perform antiviral experiment. And the route of administration and dosage capacity are also restricted. A strong virulence EV71 virus strain was selected after screening from five EV71 strains with 1-day-old suckling mice. A mouse-adapted EV71 strain with increased virulence in 12-day-old suckling mice, EV71-M5, was generated after five serial passages of the parental EV71 strain in mice. Virus titers of EV71 infected mice heart, liver, spleen, lung, kidney, small intestine, brain and muscle tissue were determined by cytopathic effect (CPE) assay. The virus used in this model is the first isolated EV71 strain in China. And 2-week-old suckling mice were used in this model. This is a supplement for the EV71 animal model in China. Establishment of this EV71 model will provide an attractive platform for anti-EV71 vaccine and drug development.
Animals ; Disease Models, Animal ; Enterovirus A, Human ; isolation & purification ; physiology ; Enterovirus Infections ; Female ; Heart ; virology ; Intestines ; virology ; Mice ; Mice, Inbred BALB C ; Muscles ; virology ; Viral Load ; Virulence

BACKGROUNDExtracellular matrix (ECM) orchestrates cell behaviour including growth, death, apoptosis, adhesion, migration, and invasion by activating several signalling pathways. Certain components of ECM, such as integrins, may act as receptors or co-receptors of enterovirus. ECM-activated gene expressions in myocardium of viral heart disease including myocarditis and partial cardiomyopathy remain elusive. This study was to investigate the expression of ECM-activated genes in myocardium of mouse with viral myocarditis.

METHODSBALB/c mice were infected with Coxsackie virus B3 (CVB3) to establish an animal model of myocarditis. Uninfected mice were also prepared and served as controls. Specific mRNA expression pattern in myocarditic mouse heart was analysed by an in-house cDNA microarray containing 8,192 genes. Overexpressed ECM genes were selected and subsequently confirmed by Northern blot analysis.

RESULTSNine ECM genes were isolated, from the array of 8,192 genes, as overexpressed genes in hearts of myocarditic mice in comparison with controls. Subsequent Northern blot analysis confirmed that four of the nine genes were highly expressed. Expression of these four genes, Fin15, ILk, Lamr1 and ADAMTS-1, has not been reported previously to be induced by Coxsackie virus.

CONCLUSIONCVB3-induced myocarditis is associated with gene expression profiles of certain ECM components.

Animals ; Blotting, Northern ; Enterovirus B, Human ; Enterovirus Infections ; metabolism ; Extracellular Matrix Proteins ; genetics ; Male ; Mice ; Mice, Inbred BALB C ; Myocarditis ; metabolism ; Myocardium ; metabolism ; Oligonucleotide Array Sequence Analysis

BACKGROUND: Nation-wide outbreak of acute hemorrhagic conjunctivitis occurred in the summer, 2002 in South Korea. We identified the causative agent of this outbreak through virus culture and molecular biological techniques. METHODS: Polymerase chain reaction (PCR) was carried out with direct conjunctival swab samples and cell culture supernatants. Conjunctival swab was done at a community based-eye clinic in Seoul, September 2002. Initial screening for adenovirus and enterovirus was performed. Nested PCR for adenovirus was done with adenovirus common primers using direct swab sample, and reverse transcription PCR (RT-PCR) for enterovirus was done with enterovirus common primers. RT-PCR with primer 188/222 for VP1 region of enterovirus was done, if initial screening test was positive. PCR product was sequenced, and homology searching, compared to prototype strains, was done for serotyping. Protease 3C region of coxsackievirus A24v was amplified and sequenced with primer D1/U2. The sequence of this region was compared to those of viral isolates, which had been obtained from several Asian outbreaks since 1970. RESULTS: Conjunctival swabs were performed in 88 patients. Thirty nine (44%) samples out of the 88 were culture positive on HeLa or MRC-5 cells. Nine (100%) out of 9 culture supernatants, randomly selected from 39 culture positve samples, were positive for coxsackievirus A24v-specific RT-PCR. Phylogenetic analysis showed that sequences from 14 culture positive supernatants, randomly selected from 39 culture positive samples, clustered into a time-related, but distinct lineage, with Asian strains. CONCLUSIONS: We identified the causative agent of the epidemic hemorrhagic conjunctivits in year 2002 as coxsackievirus A24v.
Adenoviridae ; Asian Continental Ancestry Group ; Cell Culture Techniques ; Conjunctivitis, Acute Hemorrhagic* ; Disease Outbreaks ; Enterovirus ; Enterovirus C, Human* ; Humans ; Korea* ; Mass Screening ; Polymerase Chain Reaction ; Reverse Transcription ; Seoul ; Serotyping

BACKGROUND: Nation-wide outbreak of acute hemorrhagic conjunctivitis occurred in the summer, 2002 in South Korea. We identified the causative agent of this outbreak through virus culture and molecular biological techniques. METHODS: Polymerase chain reaction (PCR) was carried out with direct conjunctival swab samples and cell culture supernatants. Conjunctival swab was done at a community based-eye clinic in Seoul, September 2002. Initial screening for adenovirus and enterovirus was performed. Nested PCR for adenovirus was done with adenovirus common primers using direct swab sample, and reverse transcription PCR (RT-PCR) for enterovirus was done with enterovirus common primers. RT-PCR with primer 188/222 for VP1 region of enterovirus was done, if initial screening test was positive. PCR product was sequenced, and homology searching, compared to prototype strains, was done for serotyping. Protease 3C region of coxsackievirus A24v was amplified and sequenced with primer D1/U2. The sequence of this region was compared to those of viral isolates, which had been obtained from several Asian outbreaks since 1970. RESULTS: Conjunctival swabs were performed in 88 patients. Thirty nine (44%) samples out of the 88 were culture positive on HeLa or MRC-5 cells. Nine (100%) out of 9 culture supernatants, randomly selected from 39 culture positve samples, were positive for coxsackievirus A24v-specific RT-PCR. Phylogenetic analysis showed that sequences from 14 culture positive supernatants, randomly selected from 39 culture positive samples, clustered into a time-related, but distinct lineage, with Asian strains. CONCLUSIONS: We identified the causative agent of the epidemic hemorrhagic conjunctivits in year 2002 as coxsackievirus A24v.
Adenoviridae ; Asian Continental Ancestry Group ; Cell Culture Techniques ; Conjunctivitis, Acute Hemorrhagic* ; Disease Outbreaks ; Enterovirus ; Enterovirus C, Human* ; Humans ; Korea* ; Mass Screening ; Polymerase Chain Reaction ; Reverse Transcription ; Seoul ; Serotyping

Human Enterovirus C group (HEV-C) includes 17 serotypes, which can not be serotype-identified by neutralization test using antiserum pool for NPEV. In order to elucidate the genotypes and molecular evolution of HEV-C in Shandong Province, We selected the strains isolated from AFP cases between 1994-2009 to perform reverse transcriptase-polymerase chain reactions (RT-PCR) by the primers specific for entire VP1 coding gene of HEV-C and sequencing. The phylogenetic tree was then constructed among these VP1 nucleotide sequences and other prototype strains. Totally 12 Shandong local strains were obtained and separated into 4 genotypes, CVA20, CVA21,CVA24 and EV 96. The homologous comparison and phylogenetic analysis showed Shandong strains were distinct from prototype strains in each genotype. This report showed that different genotype HEV-C strains spread widely in Shandong Province.
Cell Line ; China ; Enterovirus C, Human ; classification ; genetics ; Genotype ; Humans ; Molecular Sequence Data ; Phylogeny ; Reverse Transcriptase Polymerase Chain Reaction

OBJECTIVETo document ultrastructural changes of brain, spinal cord, skeletal muscle, jejunum and lung of EV71 infection mouse model, and to explore the myotropism and pathogenesis of EV71 in nervous system.

METHODSTen-day-old suckling mice were infected with EV71 strain via the intraperitoneal route. Mice with paralysis were scarified on day 4 post infection and the brain, spinal cord, skeletal muscle, jejunum and lung were sampled for transmission electron microscopy and light microscopy.

RESULTSLesions in brain were generally mild with inner chamber swelling in some of mitochondria. Myelin sheaths of medullated fibers were split with vacuolated changes. The Nissl bodies in anterior motor neurons disappeared along with mitochondria swelling, rough endoplasmic reticulum swelling and degranulation. Cytoplasm of anterior motor neurons showed cribriform appearance accompanied by neuronophagia. The bands of skeletal muscle in the infected group disappeared with degeneration and karyopyknosis in myocytes, in addition to mitochondrial swelling. Microvilli of epithelium in jejunum became loosely arranged along with formation of spiral medullary sheath structure and mitochondria swelling. Interstitial pneumonia was observed in lungs with type II pneumocyte proliferation and evacuation of the multilamellar bodies.

CONCLUSIONSEV71 infection causes severe myositis in the mouse model suggesting a strong myotropism of EV71 virus. The presence of lesions of various degrees in central nervous system and changes in anterior motor neurons may be associated with limb paralysis.

Animals ; Brain ; ultrastructure ; virology ; Disease Models, Animal ; Enterovirus A, Human ; Enterovirus Infections ; pathology ; virology ; Jejunum ; ultrastructure ; virology ; Lung ; ultrastructure ; virology ; Mice ; Mice, Inbred BALB C ; Microscopy, Electron, Transmission ; Muscle, Skeletal ; ultrastructure ; virology ; Spinal Cord ; ultrastructure ; virology