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 investigate the effect of tranilast on myocardial fibrosis in mice with viral myocarditis (VMC).

METHODSMale balb/c mice (n=72) were randomly divided into control, VMC and tranilast groups (n=24 each). In the VMC and tranilast groups, the mice were infected with Coxsackie virus B3 (CVB3) to prepare VMC model, while the control group was treated with Eagle's medium. After modeling, the tranilast group was administrated with tranilast [200 mg/(kg.d)] until the day before sampling. On days 7, 14 and 28 after CVB3 or Eagle's medium infection, heart specimens (n=8) were taken and examined after Toluidine blue staining and Nissl staining for counts of mast cells (MC), hematoxylin-eosin staining for myocardial pathological changes, and Masson staining for myocardial fibrosis. The expression of CTGF and type I collagen (Col I) in the myocardial tissue was measured by RT-PCR and Western blot. The correlations of CTGF mRNA expression with MC counts and Col I expression were analyzed.

RESULTSThe myocardial pathological changes and collagen volume fraction in the VMC group were significantly higher than in the control group at all three time points (P<0.05). Tranilast treatment significantly decreased the myocardial pathological changes and collagen volume fraction compared with the VMC group (P<0.05). The mRNA and protein expression of CTGF and Col I increased in the VMC group compared with the control group, and the increases were reduced with tranilast treatment (P<0.05). The number of MC was positively correlated to CTGF mRNA expression on the 7th day and 14th day (r=0.439, P=0.049) in the VMC group. There were positive correlations between the mRNA expression of Col I and CTGF on the 7th day and 14th day (r=0.646, P=0.007) and the 28th day (r=0.326, P=0.031).

CONCLUSIONSTranilast may inhibit the aggregation of MC and down-regulate the expression of CTGF, relieving myocardial fibrosis of mice with VMC.

Animals ; Collagen Type I ; genetics ; Connective Tissue Growth Factor ; genetics ; Coxsackievirus Infections ; drug therapy ; Enterovirus B, Human ; Fibrosis ; Male ; Mice ; Mice, Inbred BALB C ; Myocarditis ; drug therapy ; Myocardium ; pathology ; RNA, Messenger ; analysis ; ortho-Aminobenzoates ; pharmacology

OBJECTIVETo investigate the dynamic expression and role of vitamin D receptor (VDR) in the myocardium of mice with viral myocarditis (VMC).

METHODSOne hundred and twenty 4-week-old male BALB/c mice were selected and assigned into control (n=40) and experimental groups (n=80). The mice in the experimental group were injected intraperitoneally with Coxsackievirus B3 to establish the model of VMC, while the mice in the control group were injected intraperitoneally with an equal volume of DMEM solution. Fifteen mice in the experimental group and ten mice in the control group were sacrificed at 3, 7, 14, or 28 days after injection, and the myocardial specimens were obtained. The dynamic expression of VDR in the myocardium was determined by the immunohistochemical technique. The pathological changes in the myocardium were examined using hematoxylin and eosin staining.

RESULTSIn the experimental group, the mice had significantly increased expression of VDR after virus injection (P<0.01); the expression of VDR reached the peak at 7 days after injection, and then declined gradually; the expression of VDR remained high at 28 days after injection. At 3, 7, 14, and 28 days after injection, the expression of VDR in the experimental group was significantly higher than that in the control group (P<0.01). Moreover, in the experimental group, the changes in the pathological score of the myocardium were in accordance with the changes in the expression of VDR; the expression level of VDR in the myocardium was positively correlated with the pathological changes in the myocardium in the experimental group (P<0.01).

CONCLUSIONSVDR may be involved in the inflammatory-immune process in the pathogenesis of VMC.

Animals ; Coxsackievirus Infections ; metabolism ; Enterovirus B, Human ; Immunohistochemistry ; Male ; Mice ; Mice, Inbred BALB C ; Myocarditis ; metabolism ; pathology ; Myocardium ; chemistry ; pathology ; Receptors, Calcitriol ; analysis ; physiology

To identify the cause of an outbreak of acute hemorrhagic conjunctivitis (AHC) in Jiangxi (China) in 2010, 20 eye conjunctival swabs were first collected from AHC patients. Then, viruses were isola- ted and tested for human enterovirus 70, coxsackievirus A24 variant (CV-A24v) and adenovirus using the polymerase chain reaction. All CV-A24v isolates underwent sequencing of 3C and VP1 coding regions. Then, a phylogenetic tree was constructed for Jiangxi CV-A24v and worldwide CV-A24v based on,3C and VP1 regions, respectively. Ten out of 20 specimens were positive for CV-A24v, implying that the outbreak was caused by CV-A24v. The phylogenetic tree based on the 3C region showed that Jiangxi CV- A24v belonged to cluster 5 in genotype IV (GIV-C5) with strains isolated throughout the world after 2010, and were divided further into A and B lineages. Phylogenetic analyses of the VP1 region showed that all of the worldwide CV-A24v strains isolated after 2000 could be divided into five groups (1-5). Jiangxi CV-A24v was classified into group 5 and also divided further into A and B lineages upon analyses of the 3C region. These data suggested that CV-A24v causing AHC outbreaks in China in 2010 belonged to GIV-C3 and GIV-C5. At least two transmission lineages were circulated in Jiangxi in 2010. The classification of CV-A24v isolated after 2010 worldwide using the phylogenetic tree based on the VP1 region was almost consistent with that based on the 3C region and also had significant chronological clustering.
China ; epidemiology ; Conjunctivitis, Acute Hemorrhagic ; epidemiology ; virology ; Coxsackievirus Infections ; epidemiology ; virology ; Disease Outbreaks ; Enterovirus C, Human ; classification ; genetics ; isolation & purification ; Genotype ; Humans ; Molecular Sequence Data ; Phylogeny ; Viral Proteins ; genetics

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

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

OBJECTIVETo investigate the role and significance of cardiac mast cells and Toll-like receptor 4 (TLR4) in the development and progression of viral myocarditis (VMC).

METHODSForty-eight Balb/c mice were randomly divided into a control group (n=24) and a model group (n=24). Coxsackievirus B3 was intraperitoneally injected into the model group mice to establish a VMC model. In each group, cardiac tissues were collected from 8 mice at 7, 14 and 28 days after the model was established. The cardiac tissues were stained with hematoxylin and eosin as well as Masson trichrome to observe pathological changes in cardiac tissues. The number and degranulation of cardiac mast cells at each time point were measured and evaluated by toluidine blue staining and transmission electron microscopy. The mRNA and protein expression of TLR4 in cardiac tissues was measured by RT-PCR and immunohistochemistry. In the model group, the correlation between number of cardiac mast cells and mRNA expression of TLR4 at all time points was analyzed.

RESULTSThe model group had significantly higher pathological scores of cardiac tissues than the control group at all time points (P<0.05). The myocardial collagen volume fraction in the model group at 28 days was significantly higher than in the control group at all time points and higher than in the model group at 7 and 14 days (P<0.05). At each time point, the model group had a significantly increased number of mast cells (P<0.05), and significantly increased mRNA and protein expression of TLR4 (P<0.05) compared with the control group. In the model group, the number of cardiac mast cells was positively correlated with the mRNA expression of TLR4 at all time points (R²=0.877, P<0.05).

CONCLUSIONSMice with VMC have significantly increased numbers of cardiac mast cells and expression of TLR4 compared with control mice at all time points, suggesting that mast cells and TLR4 may play important roles in the inflammatory response and fibrosis of VMC.

Animals ; Coxsackievirus Infections ; immunology ; Enterovirus B, Human ; Female ; Mast Cells ; physiology ; Mice ; Mice, Inbred BALB C ; Myocarditis ; immunology ; Myocytes, Cardiac ; pathology ; Toll-Like Receptor 4 ; analysis ; genetics ; physiology

BACKGROUNDMacrophage migration inhibitory factor (MIF) is an upstream regulator in immune and inflammatory responses. However, its role in viral myocarditis remains unknown. In this study, we investigated the role of the MIF in coxsackievirus B3 (CVB3)-induced myocarditis.

METHODSMice were randomized into two groups receiving either Eagle's minimal essential medium (EMEM, control group) or virus solution (infected group). Subsets of mice in the infected group were sacrificed on days 3, 7, 14 and 28 after inoculation. Expression of MIF was detected using an enzyme-linked immunosorbent assay (ELISA), reverse transcription polymerase chain reaction and immunohistochemistry. A neutralizing antibody (Ab) to MIF was injected intraperitoneally from day 0 to 7 after inoculation. Disease severity was estimated by histopathology of the heart and by the heart weight to body weight ratio, and the interleukin-1β (IL-1β) and tumor necrosis factor α (TNF-α) in the myocardium were measured by ELISA on day 14.

RESULTSThe serum MIF concentration and expression levels of myocardial MIF mRNA and protein were significantly elevated in mice on days 7 and 14 post-infection. The survival rate was markedly higher and disease severity was obviously less in mice treated with anti-MIF Ab. Furthermore, MIF blockade significantly decreased the IL-1β and TNF-α in the myocarditic heart.

CONCLUSIONThese results demonstrate that MIF is an important naturally occurring inflammatory cytokine in CVB3-induced myocarditis, and anti-MIF Ab may lessen the inflammatory response.

Animals ; Coxsackievirus Infections ; metabolism ; pathology ; virology ; Enterovirus B, Human ; Enzyme-Linked Immunosorbent Assay ; Immunohistochemistry ; Interleukin-1beta ; metabolism ; Macrophage Migration-Inhibitory Factors ; metabolism ; Male ; Mice ; Mice, Inbred BALB C ; Myocarditis ; metabolism ; pathology ; virology ; Myocardium ; metabolism ; Reverse Transcriptase Polymerase Chain Reaction ; Tumor Necrosis Factor-alpha ; metabolism

To identify the pathogen of acute hemorrhagic conjunctivitis (AHC) in Shandong Province in 2010, eye mucous swab samples were collected from 26 patients in Qingdao and Linyi City. Real time-PCR assays for EV70, CVA24 and Adenovirus were performed on these samples. The result showed 17 samples (65.39%) were CVA24 positive while all the samples for HEV70 and Adenovirus detection were negative, which implied that CVA24 was the causative pathogen of this outbreak. A total of 10 virus strains isolated on Hep-2 cells were identified as CVA24 through VP1 amplification and nucleotide sequence analysis. The nucleotide and amino acid homologies on VP1 region among these isolates were 99.3%-100.0% and 99.5%-100.0%, respectively, and the strains aggregated together to one clade in phylogenetic tree. These results showed that the CVA24 circulating in Qingdao and Linyi City belonged to one transmission chain. Shandong CVA24s segregated into 5 different clades, and great nucleotide divergence was observed be tween AHC isolates and others.
Amino Acid Sequence ; China ; epidemiology ; Conjunctivitis, Acute Hemorrhagic ; epidemiology ; virology ; Enterovirus C, Human ; chemistry ; classification ; genetics ; isolation & purification ; Genetic Variation ; Humans ; Molecular Sequence Data ; Phylogeny ; Sequence Homology, Amino Acid ; Viral Proteins ; chemistry ; genetics

To evaluate the effects of lentivirus-delivered short hairpin RNA (shRNA) on CVB3 infection in an animal model by RNA interference technique, we constructed a recombinant lentivirus expressing shRNA-3753 against the viral genome region 3753-3771, then transduced Lenti-sh3753 into mice infected with CVB3. We evaluated the antiviral ability of lenti-sh3753 by cytopathic effect (CPE), viral plaque assay and histological analysis of mice hearts. The results showed that Lenti-sh3753 exhibited a significant protective effect on cell viability and reduction of viral titers in supernatant of cell culture by specific inhibition on viral replication. Lenti-sh3753 also prolonged the mice survival and limited the viral production in mice hearts. These data proposed that Lenti-sh3753 can effectively inhibit CVB3 infection in a coxsackievirus-induced myocarditis model, suggesting its potential role in prevention and therapy of viral diseases.
Animals ; Coxsackievirus Infections ; drug therapy ; virology ; Down-Regulation ; Enterovirus B, Human ; genetics ; physiology ; Humans ; Male ; Mice ; Mice, Inbred BALB C ; Myocarditis ; drug therapy ; virology ; RNA Interference ; RNA, Small Interfering ; genetics ; therapeutic use ; RNA, Viral ; genetics ; Virus Replication