[Vascular endothelial growth factor induces inflammatory injury of pancreatic tissue by activating autophagy in hyperlipidemic acute pancreatitis rats].

This study was to investigate the changes of autophagy in pancreatic tissue cells from hyperlipidemic acute pancreatitis (HLAP) rats and the molecular mechanism of autophagy to induce inflammatory injury in pancreatic tissue cells. Male Sprague Dawley (SD) rats were intraperitoneally injected with caerulein to establish acute pancreatitis (AP) model and then given a high fat diet to further prepare HLAP model. The HLAP rats were treated with autophagy inducer rapamycin or inhibitor 3-methyladenine. Pancreatic acinar (AR42J) cells were treated with caerulein to establish HLAP cell model. The HLAP cell model were treated with rapamycin or transfected with vascular endothelial growth factor (VEGF) siRNA. The inflammatory factors in serum and cell culture supernatant were detected by ELISA method. The histopathological changes of pancreatic tissue were observed by HE staining. The changes of ultrastructure and autophagy in pancreatic tissue were observed by electron microscopy. The expression levels of Beclin-1, microtubule- associated protein light chain 3-II (LC3-II), mammalian target of rapamycin complex 1 (mTORC1), and VEGF were measured by immunohistochemistry and Western blot. The results showed that, compared with control group, the autophagy levels and inflammatory injury of pancreatic tissue cells from HLAP model rats were obviously increased, and these changes were aggravated by rapamycin treatment, but alleviated by 3-methyladenine treatment. In HLAP cell model, rapamycin aggravated the autophagy levels and inflammatory injury, whereas VEGF siRNA transfection increased mTORC1 protein expression, thus alleviating the autophagy and inflammatory injury of HLAP cell model. These results suggest that VEGF-induced autophagy plays a key role in HLAP pancreatic tissue cell injury, and interference with VEGF-mTORC1 pathway can reduce the autophagy levels and alleviate the inflammatory injury. The present study provides a new target for prevention and treatment of HLAP.

The efficacy of tolvaptan for heart failure in chronic kidney disease: A protocol for systematic review and meta-analysis.

BACKGROUND: Approximately half of people with heart failure have chronic kidney disease (CKD). Tolvaptan is reported to be effective in treating heart failure. However, the safety and efficacy of its use in patients with CKD is uncertain. In this study, we conducted a protocol for systematic review and meta-analysis to investigate the efficacy and safety of tolvaptan on patients with heart failure and CKD. METHODS: This study protocol has been registered in the PROSPERO and the registration number is CRD42022368148. The consent of this protocol report is based on the Preferred Reporting Items for Systematic Review and Meta-Analysis Protocols (PRISMA-P) 2015 statement guidelines. We will include randomized controlled trials related to tolvaptan in patients with heart failure and CKD. Two research members will electronically and independently search 4 English databases (EMBASE, PubMed, National Guideline Clearinghouse, and Cochrane Central Register of Controlled Trials) and 4 Chinese databases (Chinese Biomedical Literature Database, Chinese National Knowledge Infrastructure, Wanfang Database, and VIP Database) from their inception to November 2022. The risk of bias in each included study will be assessed utilizing the Cochrane Collaboration's risk of bias tool. All statistical analyses will be conducted using the software program Review Manager version 5.3. RESULTS: The results of this systematic review will be published in a peer-reviewed journal. CONCLUSION: This review can provide convincing evidence to help clinicians make decisions when dealing with heart failure and CKD.

Comprehensive analysis of the correlation between GSTM1 and tumor immunity in colon cancer.

Background: Glutathione S-transferase mu 1 (GSTM1) is one of the major glutathione conjugation enzymes. Its expression and activity have been suggested to correlate with the occurrence of colon cancer; however, the role of GSTM1 in tumor immunity remains unclear. Methods: Relevant data downloaded from The Cancer Genome Atlas (TCGA), Clinical Proteomic Tumor Analysis Consortium (CPTAC), and Human Protein Atlas (HPA) was used to perform a multi-dimensional expression analysis of GSTM1 in colon adenocarcinoma (COAD). The correlation between GSTM1 and tumor immunity was analyzed with multiple online tools. Then protein-protein interaction (PPI) network and functional enrichment analyses of GSTM1-associated immunomodulators were performed. Further, we developed the Cox regression model based on the GSTM1-related immunomodulators. Finally, a GSTM1-based clinical nomogram and a calibration curve was established to predict the probability and accuracy of long-term survival. Result: GSTM1 was significantly downregulated in COAD versus normal tissues. Infiltration levels of B cells, CD8+ T cells, and dendritic cells were closely correlated to GSTM1 gene copy number deletion, and GSTM1 expression levels in COAD positively correlated with dendritic cell, B cell, neutrophil, and macrophage infiltration. Functional enrichment analysis indicated 36 GSTM1-related immunomodulators are involved in immune-related pathways of regulating T cell activation and lymphocytic activation. A 2-gene prognostic risk signature based on the 36 GSTM1-related immunomodulators was built using the Cox regression model, and the risk signature in combination with stage had an area under the curve (AUC) value of 0.747 by the receiver operating characteristic method. patients with higher risk scores-calculated based on 2 gene prognostic risk characteristics and further identified as an independent prognostic factor-were associated with worse survival using the Kaplan-Meier analysis. Together, the clinical nomogram and calibration curve based on GSTM1 suggested a good prediction accuracy for long-term survival probability. Conclusions: Our study provided evidence supporting the significant role of GSTM1 in COAD immunity and suggests GSTM1 as a potential novel target for COAD immunotherapy.

An adult gerbil model for evaluating potential coxsackievirus A16 vaccine candidates.

A suitable animal model of CVA16 infection is crucial in order to understand its pathogenesis and to help develop antiviral vaccines or screen therapeutic drugs. The neonatal mouse model has a short sensitivity period to CA16 infection, which is a major limitation. In this study, we demonstrate that adult (60-day-old) gerbils are susceptible to CVA16 infection at high doses (108.0 TCID50). A clinical isolate strain of CVA16 was inoculated intraperitoneally into adult gerbils, which subsequently developed significant clinical symptoms, including hind limb weakness, paralysis of one or both hind limbs, tremors, and eventual death from neurological disorders. Real-time RT-PCR revealed that viral loads in the spinal cord and brainstem were higher than those in other organs/tissues. Histopathological changes, such as neuronal degeneration, neuronal loss, and neuronophagia, were observed in the spinal cord, brainstem, and heart muscle, along with necrotizing myositis. Gerbils receiving both prime and boost immunizations of alum adjuvant inactivated vaccine exhibited no clinical signs of disease or mortality following challenge by CVA16, whereas 80% of control animals showed obvious clinical signs, including slowness, paralysis of one or both hind limbs, and eventual death, suggesting that the CVA16 vaccine can fully protect gerbils against CVA16 challenge. These results demonstrate that an adult gerbil model provides us with a useful tool for studying the pathogenesis and evaluating antiviral reagents of CVA16 infection. The development of this animal model would also be conducive to screening promising CVA16 vaccine candidates as well as further vaccination evaluation.

Risk Factors and Preventions of Breast Cancer.

Breast cancer is the second leading cause of cancer deaths among women. The development of breast cancer is a multi-step process involving multiple cell types, and its prevention remains challenging in the world. Early diagnosis of breast cancer is one of the best approaches to prevent this disease. In some developed countries, the 5-year relative survival rate of breast cancer patients is above 80% due to early prevention. In the recent decade, great progress has been made in the understanding of breast cancer as well as in the development of preventative methods. The pathogenesis and tumor drug-resistant mechanisms are revealed by discovering breast cancer stem cells, and many genes are found related to breast cancer. Currently, people have more drug options for the chemoprevention of breast cancer, while biological prevention has been recently developed to improve patients' quality of life. In this review, we will summarize key studies of pathogenesis, related genes, risk factors and preventative methods on breast cancer over the past years. These findings represent a small step in the long fight against breast cancer.

[Effects of acute hypoxia on telomere length of rat gastric mucosa tissue and underlying mechanism].

The present study was aimed to investigate the effect of acute hypoxia on telomere length of rat gastric mucosa tissue and possible mechanism. Forty male Wistar rats were randomly divided into control group (resided in Lanzhou, 1 500 m) and experimental group (hypoxia chamber, 5 000 m). The experimental group was further divided into 3 subgroups and exposed to hypoxia for 1, 3, 7 d (n = 10), respectively. The morphological changes of the gastric mucosa tissue were observed by HE staining. By means of real-time PCR, ELISA and chemical immunofluorescence methods, the telomere length, the mRNA and protein levels of telomerase reverse transcriptase (TERT), hypoxia-inducible factor 1α (HIF-1α) and HIF-2α, and reactive oxygen species (ROS) level in gastric mucosa tissue were measured, respectively. The results showed that, with the extension of hypoxia-exposure time, the injury in gastric mucosa cells progressively became worse, and telomere length was increased gradually, along with intracellular ROS generation. The changes of TERT and HIF-1α expressions induced by acute hypoxia were in the same trend as that of telomere length. There were positive correlations between TERT mRNA expression and telomere length and between TERT and HIF-1α expressions, but not between TERT and HIF-2α mRNA expressions. These results suggest that under acute severe hypoxia environment, ROS could damage the gastric mucosa tissue cells, meanwhile the expressions of TERT and telomerase activity may be up-regulated by HIF-1α, which can elongate the telomere length and protect gastric mucosa tissue against fatal injury.

Coxsackievirus A16 induced neurological disorders in young gerbils which could serve as a new animal model for vaccine evaluation.

Coxsackievirus A16 (CA16) is one of the major pathogens associated with human hand, foot, and mouth disease (HFMD) in the Asia-pacific region. Although CA16 infections are generally mild, severe neurological manifestations or even death has been reported. Studies on CA16 pathogenesis and vaccine development are severely hampered because the small animal models that are currently available show major limitations. In this study, gerbils (Meriones unguiculatus) were investigated for their suitability as an animal model to study CA16 pathogenesis and vaccine development. Our results showed that gerbils up to the age of 21 days were fully susceptible to CA16 and all died within five days post-infection. CA16 showed a tropism towards the skeletal muscle, spinal cord and brainstem of gerbils, and severe lesions, including necrosis, were observed. In addition, an inactivated CA16 whole-virus vaccine administrated to gerbils was able to provide full protection to the gerbils against lethal doses of CA16 strains. These results demonstrate that gerbils are a suitable animal model to study CA16 infection and vaccine development.

Improvements on high-precision measurement of bromine isotope ratios by multicollector inductively coupled plasma mass spectrometry.

A new, feasible procedure for high-precision bromine isotope analysis using multicollector inductively coupled plasma mass spectrometry (MC-ICP-MS) is described. With a combination of HR mass resolution mode and accurate optimization of the Zoom Optics parameters (Focus Quad: -1.30; Zoom Quad: 0.00), the challenging problem of the isobaric interferences ((40)Ar(38)ArH(+) and (40)Ar(40)ArH(+)) in the measurement of bromine isotopes ((79)Br(+), (81)Br(+)) has been effectively solved. The external reproducibility of the measured (81)Br/(79)Br ratios in the selected standard reference materials ranged from ±0.03‰ to ±0.14‰, which is superior to or equivalent to the best results from previous contributions. The effect of counter cations on the Br(+) signal intensity and the instrumental-induced mass bias was evaluated as the loss of HBr aerosol in nebulizer and potential diffusive isotope fractionations.

[An effective method for improving the imaging spatial resolution of terahertz time domain spectroscopy system].

Terahertz radiation is an electromagnetic radiation in the range between millimeter waves and far infrared. Due to its low energy and non-ionizing characters, THz pulse imaging emerges as a novel tool in many fields, such as material, chemical, biological medicine, and food safety. Limited spatial resolution is a significant restricting factor of terahertz imaging technology. Near field imaging method was proposed to improve the spatial resolution of terahertz system. Submillimeter scale's spauial resolution can be achieved if the income source size is smaller than the wawelength of the incoming source and the source is very close to the sample. But many changes were needed to the traditional terahertz time domain spectroscopy system, and it's very complex to analyze sample's physical parameters through the terahertz signal. A method of inserting a pinhole upstream to the sample was first proposed in this article to improve the spatial resolution of traditional terahertz time domain spectroscopy system. The measured spatial resolution of terahertz time domain spectroscopy system by knife edge method can achieve spatial resolution curves. The moving stage distance between 10 % and 90 Yo of the maximum signals respectively was defined as the, spatial resolution of the system. Imaging spatial resolution of traditional terahertz time domain spectroscopy system was improved dramatically after inserted a pinhole with diameter 0. 5 mm, 2 mm upstream to the sample. Experimental results show that the spatial resolution has been improved from 1. 276 mm to 0. 774 mm, with the increment about 39 %. Though this simple method, the spatial resolution of traditional terahertz time domain spectroscopy system was increased from millimeter scale to submillimeter scale. A pinhole with diameter 1 mm on a polyethylene plate was taken as sample, to terahertz imaging study. The traditional terahertz time domain spectroscopy system and pinhole inserted terahertz time domain spectroscopy system were applied in the imaging experiment respectively. The relative THz-power loss imaging of samples were use in this article. This method generally delivers the best signal to noise ratio in loss images, dispersion effects are cancelled. Terahertz imaging results show that the sample's boundary was more distinct after inserting the pinhole in front of, sample. The results also conform that inserting pinhole in front of sample can improve the imaging spatial resolution effectively. The theoretical analyses of the method which improve the spatial resolution by inserting a pinhole in front of sample were given in this article. The analyses also indicate that the smaller the pinhole size, the longer spatial coherence length of the system, the better spatial resolution of the system. At the same time the terahertz signal will be reduced accordingly. All the experimental results and theoretical analyses indicate that the method of inserting a pinhole in front of sample can improve the spatial resolution of traditional terahertz time domain spectroscopy system effectively, and it will further expand the application of terahertz imaging technology.

Enterovirus 71 infection causes severe pulmonary lesions in gerbils, meriones unguiculatus, which can be prevented by passive immunization with specific antisera.

Neurogenic pulmonary edema caused by severe brainstem encephalitis is the leading cause of death in young children infected by Enterovirus 71 (EV71). However, no pulmonary lesions have been found in EV71-infected transgenic or non-transgenic mouse models. Development of a suitable animal model is important for studying EV71 pathogenesis and assessing effect of therapeutic approaches. We had found neurological disorders in EV71-induced young gerbils previously. Here, we report severe pulmonary lesions characterized with pulmonary congestion and hemorrhage in a gerbil model for EV71 infection. In the EV71-infected gerbils, six 21-day-old or younger gerbils presented with a sudden onset of symptoms and rapid illness progression after inoculation with 1×105.5 TCID50 of EV71 via intraperitoneal (IP) or intramuscular (IM) route. Respiratory symptoms were observed along with interstitial pneumonia, pulmonary congestion and extensive lung hemorrhage could be detected in the lung tissues by histopathological examination. EV71 viral titer was found to be peak at late stages of infection. EV71-induced pulmonary lesions, together with severe neurological disorders were also observed in gerbils, accurately mimicking the disease process in EV71-infected patients. Passive transfer with immune sera from EV71 infected adult gerbils with a neutralizing antibody (GMT=89) prevented severe pulmonary lesion formation after lethal EV71 challenge. These results establish this gerbil model as a useful platform for studying the pathogenesis of EV71-induced pulmonary lesions, immunotherapy and antiviral drugs.

Separation and purification of flavonoid from Taxus remainder extracts free of taxoids using polystyrene and polyamide resin.

An efficient separation process of flavonoid from Taxus wallichiana var. mairei remainder extracts free of taxoids was developed in this study. AB-8 macroporous resin and polyamide resin offered the fine adsorption capacity, and its adsorption rate at 30°C fitted well to the Langmuir and Freundich isotherms. Resin dynamic adsorption and desorption experiments were conducted to optimize the separation process of total flavonoids from T. wallichiana var. mairei remainder extracts free of taxoids. The optimum parameters for adsorption by AB-8 resin were as follows: (1) the concentration of flavonoids in a sample solution of 5.61 mg/mL with a processing volume of 2 bed volume (BV) (60 mL); (2) for desorption, ethanol-water (80:20, v/v), with 6 BV as an eluent at a flow rate of 2 BV/h. After a one-run treatment with AB-8 resin, the content of flavonoids was increased 5.10-fold from 4.05 to 20.65%. The optimum parameters for adsorption by polyamide resin were as follows: processing volume of 2 BV (30 mL); for desorption, ethanol-water (70:30, v/v), with 8 BV as an eluent at a flow rate of 2 BV/h. After one-run treatment with polyamide resin, the content of total flavonoids increased from 20.65 to 65.21%. The method will provide a potential approach for large-scale separation and purification of flavonoid for its wide pharmaceutical use.

Atom probe tomography simulations and density functional theory calculations of bonding energies in Cu3Au.

In this article the Cu-Au binding energy in Cu3Au is determined by comparing experimental atom probe tomography (APT) results to simulations. The resulting bonding energy is supported by density functional theory calculations. The APT simulations are based on the Müller-Schottky equation, which is modified to include different atomic neighborhoods and their characteristic bonds. The local environment is considered up to the fifth next nearest neighbors. To compare the experimental with simulated APT data, the AtomVicinity algorithm, which provides statistical information about the positions of the neighboring atoms, is applied. The quality of this information is influenced by the field evaporation behavior of the different species, which is connected to the bonding energies.

Enterovirus 71-induced neurological disorders in young gerbils, Meriones unguiculatus: development and application of a neurological disease model.

A reliable disease model mimicking Enterovirus 71 (EV71) infection in humans is essential for understanding pathogenesis and for developing a safe and effective vaccine. Commonly used rodent models including mouse or rat models are not suitable for vaccine evaluation because the rodents are resistant to EV71 infection after they reach the age of 6 days. In this study, 21-day-old gerbils inoculated intraperitoneally (IP) with a non mouse-adapted EV71 strain developed neurological lesion-related signs including hind limb paralysis, slowness, ataxia and lethargy similar to those of central nervous system (CNS) infection of EV71 in humans. The infected gerbils eventually died of the neurological lesions and EV71 could be isolated from lung, liver, spleen, kidney, heart, spinal cord, brain cortex, brainstem and skeletal muscle. Significantly high virus replication was detected in spinal cord, brainstem and skeletal muscle by cellular analysis, real-time quantitative PCR (RT-PCR) and immunohistochemical staining. Histopathologic changes such as neuronal degeneration, neuronal loss and neuronophagia were observed in spinal cord, brain cortex, brainstem, and skeletal muscle along with necrotizing myositis and splenic atrophy. Gerbils that received two doses of inactive whole-virus vaccine showed no EV71-specific symptoms after challenged with EV71. In contrast, gerbils that received mock vaccination died of EV71-induced neuropathology after challenged with EV71. The result indicates that gerbils can serve as a reliable disease model for evaluating safety and efficacy of EV71 vaccine.

[Genotype of hantavirus in Zhejiang province].

OBJECTIVE: To identify the genotype and clades of hantavirus (HV) in Zhejiang province. METHODS: The partial S and M segment of the HV in Zhejiang province were amplified with RT-PCR using genotype-specific primers, and then were sequenced and compared with other known hantaviruses. RESULTS: The genotype of 11 strains were HTNV and other 7 strains were SEOV by homology and phylogenesis analysis, yet the clade distribution was significantly different among foci of Zhejiang with 5 clades of HTNV and 3 clades of SEOV. There also existed special clade of HTNV named ZNB-1, ZNB-2, A3 and of SEOV named Gou3, ZJ5. The homology of M segments of ZNB-1 and ZNB-2 with other HTNV clades were 69.7%-74.0% except Nc167, A3 with other HTNV clades were 73.6%-76.3% except B78. CONCLUSION: Zhejiang province is co-circulating with HTN and SEO. Say the least of the clades are 5 of HTNV and 3 of SEOV and there also existed special clade of HTNV and SEOV.

[The expression and bioactivity of S gene in Vero-E6 cells using baculovirus vector].

OBJECTIVE: The S gene of a Hanta Virus (HV) Z10 strain was cloned into a baculovirus shuttle bacmid pDual-CMV contained a CMV promoter to generated a recombinant baculovirus BAC-pDual-CMV-HVS, then the recombinant baculovirus was transfected into Vero-E6 cell. The cells with recombinant baculovirus were applied to the detection of HV antiserum. METHODS: To generate the recombinant baculovirus BAC-pDual-CMV-HVS, the sequence of CMV promoter was obtained from the plasmid pEGFP-N1 by PCR, and subsequently cloned to the baculovirus shuttle bacmid pFastBacDUAL resulting the recombinant plasmid pDual-CMV. Then the sequence of HV-S gene was inserted to the plasmid pDual-CMV, to generate the plasmid pDual-CMV-HVS. Plasmid pDual-CMV-HVS was transformed into the DH10BAC competent cells to get the recombinant baculovirus BAC-pDual-CMV-HVS. The antigen substrate slides were made by transfecting the recombinant virus into Vero-E6 cells. RESULTS: The plasmid pDual-CMV-HVS was verified by sequencing. The recombinant virus BAC-pDual-CMV-HVS was generated according to the protocol of the baculovirus and transfected into Vero-E6 cells. The expression of the HV-S gene was verified by positive HV antiserum. CONCLUSION: [corrected] The recombinant virus were successfully generated and applied to prepare the antigen substrate slides. The antigen substrate slides was conveniently prepared without special equipments, and can be used to detect the antiserum of HV virus.

Preliminary experimental research on the mechanism of liver bile secretion stimulated by peppermint oil.

OBJECTIVE: To investigate the choleretic effect and molecular mechanisms of action of peppermint oil (PO), the main component of Danshu capsules (Sichuan Jishengtang Pharmaceutical Co., Ltd., Pengzhou, Sichuan Province, China). METHODS: Bile secretion was measured by biliary drainage in rats. Total bile acids, total cholesterol and bilirubin in bile were determined. Cholesterol 7α-hydroxylase (CYP7A1), and farnesoid X receptor (FXR) messenger ribonucleic acid (mRNA) levels were assessed in HepG2 cells (a human hepatocellular carcinoma cell line) by reverse transcription polymerase chain reaction (RT-PCR). RESULTS: PO significantly promoted bile and bile acid secretion in rats. It also increased bile acid efflux and decreased cholesterol levels (P < 0.01) in bile. In HepG2 cells the mRNA levels of CYP7A1 and FXR were significantly upregulated after treatment with PO. CONCLUSIONS: PO stimulates bile fluid secretion and thus has a choleretic effect. PO might play a role in upregulating CYP7A1 and FXR mRNA levels, suggesting that the molecular mechanisms are related to gene expression involved in bile acid synthesis.

[THz time-domain spectroscopic study of composites filled with different carbon black].

Composites were prepared by filling high density polyethylene (HDPE) with acetylene black (AC-CB) and high-structure CB (HG-CB), respectively. Optical properties of the composites were characterized with terahertz time-domain spectroscopy (THz-TDS). It was found that as frequency increases the absorption coefficients of the composites increase whereas the refractive indexes decrease. Both the absorption coefficient and refractive index increase with increasing the particle concentration. The HG-CB filled composites have larger absorption coefficient but smaller refractive index compared with that of the AC-CB composites at the same particle concentration. These phenomena are related to the different particulate structures and aggregate structures of the CB particles. Assuming that the dielectric loss in THz frequency range is mainly attributed to the electron transport within the conductive clusters and the interfacial polarization of HDPE, the information of relaxation time and relaxation strength was obtained through fitting the experimental results to two-Debye theory of dipole relaxation.

[Molecular evolution analysis of hantaviruses in Zhejiang Province].

In order to analyze the molecular epidemiology of Hantavirus (HV) in Zhejiang Province, the complete M and S genome sequences of 12 HV strains from different hosts and locations in Zhejiang Province of China during the period of 1981-2007 were analyzed on genetic evolution by DNAstar and MEGA 4.0 software in this research. Phylogenetic analyses revealed that HTN and SEO strains were co-circulating in Zhejiang Province, and the difference in sequence similarity and the phylogeny was closely related to the isolated regions, but had no distinct relationship with the isolate year and the host, indicating a relationship between epidemiology of HFRS and the distribution region, especially in HTNV. The isolates in the same region could be assigned in same or near phylogenetic clade sharing high sequence similarity. Interestingly, the Gou3 strain and ZJ5 strain isolated from Jiande region in Zhejiang Province formed a distinct phylogenetic lineage in SEOV clade, and different from the other SEOV variants outside China. We believed that the special SEOV variants were distributed in Jiande region.

[Development and evaluation of TaqMan-based one-step reverse transcription-polymerase chain reaction assay for the detection of Japanese encephalitis virus].

OBJECTIVE: To establish a TaqMan based real-time reverse transcription-polymerase chain reaction (RT-PCR) assay for the detection of Japanese encephalitis virus. METHODS: The gene sequences of Japanese encephalitis virus downloaded from the GenBank was aligned, using the biologic software. Specific primers and probes were designed in the conserved region of the C gene for Japanese encephalitis virus. The real-time RT-PCR reactive condition was optimized and the sensitivity, specificity and the stability of the assay were evaluated. Mosquitoes collected from Zhejiang province were detected by this assay. RESULTS: Mg2+, primer and probe were optimized at 5 mmol/L, 0.2 micromol/L and 0.1 micromol/L respectively. The specificity of the assay was high and there were no cross reactions with dengue virus, rabies virus, seoul virus or hantan virus. The detection limits of the assay was 0.1 TCID50. Results from preliminary application showed that TaqMan RT-PCR for Japanese encephalitis virus was sensitive, easier and faster to perform the process of traditional virus isolation and identification. It took only three hours to extract viral RNA and perform the real-time RT-PCR. CONCLUSION: This TaqMan-based one-step RT-PCR assay was a quick, sensitive and specific tool for molecular diagnosis of Japanese encephalitis virus.