CD11b maintains West Nile virus replication through modulation of immune response in human neuroblastoma cells.

BACKGROUND: West Nile virus (WNV) is a rapidly spreading mosquito-borne virus accounted for neuroinvasive diseases. An insight into WNV-host factors interaction is necessary for development of therapeutic approaches against WNV infection. CD11b has key biological functions and been identified as a therapeutic target for several human diseases. The purpose of this study was to determine whether CD11b was implicated in WNV infection. METHODS: SH-SY5Y cells with and without MEK1/2 inhibitor U0126 or AKT inhibitor MK-2206 treatment were infected with WNV. CD11b mRNA levels were assessed by real-time PCR. WNV replication and expression of stress (ATF6 and CHOP), pro-inflammatory (TNF-α), and antiviral (IFN-α, IFN-ß, and IFN-γ) factors were evaluated in WNV-infected SH-SY5Y cells with CD11b siRNA transfection. Cell viability was determined by MTS assay. RESULTS: CD11b mRNA expression was remarkably up-regulated by WNV in a time-dependent manner. U0126 but not MK-2206 treatment reduced the CD11b induction by WNV. CD11b knockdown significantly decreased WNV replication and protected the infected cells. CD11b knockdown markedly increased TNF-α, IFN-α, IFN-ß, and IFN-γ mRNA expression induced by WNV. ATF6 mRNA expression was reduced upon CD11b knockdown following WNV infection. CONCLUSION: These results demonstrate that CD11b is involved in maintaining WNV replication and modulating inflammatory as well as antiviral immune response, highlighting the potential of CD11b as a target for therapeutics for WNV infection.

Minocycline Inhibits Tick-Borne Encephalitis Virus and Protects Infected Cells via Multiple Pathways.

Tick-borne Encephalitis (TBE) is a zoonotic disease caused by the Tick-borne Encephalitis virus (TBEV), which affects the central nervous system of both humans and animals. Currently, there is no specific therapy for patients with TBE, with symptomatic treatment being the primary approach. In this study, the effects of minocycline (MIN), which is a kind of tetracycline antibiotic, on TBEV propagation and cellular protection in TBEV-infected cell lines were evaluated. Indirect immunofluorescence, virus titers, and RT-qPCR results showed that 48 h post-treatment with MIN, TBEV replication was significantly inhibited in a dose-dependent manner. In addition, the inhibitory effect of MIN on different TBEV multiplicities of infection (MOIs) in Vero cells was studied. Furthermore, the transcriptomic analysis and RT-qPCR results indicate that after incubation with MIN, the levels of TBEV and CALML4 were decreased, whereas the levels of calcium channel receptors, such as RYR2 and SNAP25, were significantly increased. MIN also regulated MAPK-ERK-related factors, including FGF2, PDGFRA, PLCB2, and p-ERK, and inhibited inflammatory responses. These data indicate that administering MIN to TBEV-infected cells can reduce the TBEV level, regulate calcium signaling pathway-associated proteins, and inhibit the MAPK-ERK signaling pathway and inflammatory responses. This research offers innovative strategies for the advancement of anti-TBEV therapy.

Inhibition of tick-borne encephalitis virus in cell cultures by ribavirin.

Tick-borne encephalitis virus (TBEV) belonging to arboviruses is a major member of zoonotic pathogens. TBEV infection causes severe human encephalitis without specific antiviral drugs. Due to its use of antiviral drug against a wide range of viruses, we investigated antiviral effect of ribavirin against TBEV in susceptible human cell lines A549 and SH-SY5Y. Ribavirin displayed minor cytotoxicity on multiple cell lines. Ribavirin obviously impaired TBEV replication and protected the infected cells from cytopathic effect. Importantly, ribavirin markedly inhibited TBEV propagation, as evidenced by impairment of TBEV production and viral RNA replication. Treatment with ribavirin (co-treatment and post-treatment) led to a dose-dependent reduction in TBEV titers as well as the viral RNA levels. Antiviral protein myxovirus resistance A mRNA expression was significantly up-regulated and signal transducer and activator of transcription 3 was activated in TBEV-infected A549 cells upon the ribavirin treatment. Induction of inflammatory cytokine tumor necrosis factor alpha by TBEV was decreased in A549 cells with the treatment of ribavirin, whereas interleukin 1 beta release appeared to be unaffected. These results suggest that ribavirin might represent a promising safe and effective antiviral drug against TBEV.

Handheld NIR-to-NIR Platform for on-site evaluating protective neutralizing antibody against SARS-CoV-2 ancestral strain and Omicron variant after vaccination or infection.

Lateral flow assays (LFAs) are promising points-of-care tests, playing a vital role in diseases screening, diagnosis and surveillance. However, development of portable, cheap, and smart LFAs platform for sensitive and accurate quantification of disease biomarkers in complex media is challenging. Here, a cheap handheld device was developed to realize on-site detection of disease biomarkers by Nd3+/Yb3+ co-doped near-infrared (NIR)-to-NIR downconversion nanoparticles (DCNPs) based LFA. Its sensitivity is at least 8-fold higher for detecting NIR light signal from Nd3+/Yb3+ co-doped nanoparticles than conventional expensive InGaAs camera based detection platform. Additionally, we enhance NIR quantum yield of Nd3+/Yb3+ co-doped nanoparticles up to 35.5% via simultaneous high dopant of sensitizer ions Nd3+ and emitter ions Yb3+. Combination of NIR-to-NIR handheld detection device and ultra-bright NIR emitting NaNbF4:Yb60%@NaLuF4 nanoparticle probe allows the detection sensitivity of SARS-CoV-2 ancestral strain and Omicron variants specific neutralizing antibodies LFA up to the level of commercial enzyme linked immunosorbent assay kit. Furthermore, by this robust method, enhanced neutralizing antibodies against SARS-CoV-2 ancestral strain and Omicron variants are observed in healthy participants with Ad5-nCoV booster on top of two doses of inactivated vaccine. This NIR-to-NIR handheld platform provides a promising strategy for on-site evaluating protective humoral immunity after SARS-CoV-2 vaccination or infection.

A New Auto-RPA-Fluorescence Detection Platform for SARS-CoV-2.

OBJECTIVE: The outbreak of COVID-19 caused by SARS-CoV-2 has led to a serious worldwide pandemic. Quantitative reverse transcription-polymerase chain reaction (qRT-PCR)-based methods were recommended for routine detection of SARS-CoV-2 RNA. Because the reaction time and analytical sensitivity of qRT-PCR limits the diagnosis of SARS-CoV-2, development of a quick process of SARS-CoV-2 detection technology with high analytical sensitivity remains urgent. METHODS: We combined isothermal amplification and fluorescence detection technology to develop a new auto-recombinase polymerase amplification (RPA)-fluorescence platform that could be used in the diagnosis of SARS-CoV-2. RESULTS: By optimization of primers and probes, the RPA platform could detect SARS-CoV-2 nucleotides within 15 min. The limits of detection and specificity of the auto-RPA-fluorescence platform were 5 copies/µL and 100%, respectively. The accuracy of detection of the auto-RPA-fluorescence platform in the 16 positive samples was 100%. CONCLUSION: The RPA platform is a potential technology for the diagnosis of SARS-CoV-2 infection.

Establishment of qRT-PCR for absolute quantitative detection of Chikungunya virus / 中国热带医学

@#Abstract: Objective　To develop a real-time fluorescent quantitative RT-PCR (qRT-PCR) method for qualitative and quantitative Chikungunya virus (CHIKV) analysis. Methods Based on the systematic analysis of the genomic sequences of Chikungunya and its related arboviruses, the specific nucleic acid sequences for Chikungunya virus were screened and identified, and then the primers and TaqMan probe were designed. Meanwhile, the human GAPDH gene was used as an internal reference. The reaction system for qRT-PCR was systematically optimized by L9(34) orthogonal design, and a rapid detection method for Chikungunya by qRT-PCR based on TaqMan probe methods was established. The sensitivity, specificity, reproducibility, and coverage of the established method were analyzed in detail. The standard curve was made, and the absolute quantitative method was established using the cloned nucleic acid fragments as positive samples. Results　A real-time fluorescent quantitative RT-PCR assay was developed for the qualitative and quantitative analysis of Chikungunya virus. The reaction system included Chikungunya virus and reference internal gene specific primers and probe, RT/Taq enzyme mixture, reaction buffer, and negative and positive reference. The established method obtained positive results with the ROSS strain of ECSA subtype, LR2006 strain of IOL branch, 181/25 strain of Asian type and Dongguan 2010 epidemic strains of Chikungunya virus, but there was no cross-reaction with other 18 arboviruses belonging to Flaviviruses, Alphaviruses and Bunyavirus. The minimum detection limit of the established method was 5.80 copies/mL, and a linear relationship was observed between the amount of input plasmid DNA and fluorescence signal value over a range of 5.80×102 copies/mL to 5.80×1010 copies/mL, and the correlation coefficient was 0.999 5. The qRT-PCR amplification efficiency was 91%, and the intra-assay variations and inter-assay variations were 0.01-0.07 and 0.03-0.11, respectively. Conclusions　The TaqMan qRT-PCR method developed in this study can qualitatively and quantitatively detect Chikungunya virus rapidly with specificity and sensitivity, providing a technical method for the prevention and control of this viral disease.

Identification of clinical candidates against West Nile virus by activity screening in vitro and effect evaluation in vivo.

The West Nile virus (WNV) is a member of the flavivirus and is known to cause encephalitis. There is currently no specific treatment for WNV infection. Repurposing of clinically approved drugs appeared promising for rapidly identifying effective, safe, and readily available candidates for antiviral drugs. Here, we screened the small-molecule compounds with anti-WNV activity from 978 Food Drug Administration-approved drugs. Four compounds, including cilnidipine, mycophenolate mofetil, nitazoxanide, and teriflunomide, were found to efficiently abrogate WNV infection in Vero cells and human neuroblastoma SH-SY5Y cells. The four compounds also exert broad-spectrum antiviral activity against the Zika virus, Japanese encephalitis virus, yellow fever virus, tick-borne encephalitis virus, and chikungunya virus. Furthermore, nitazoxanide (a synthetic benzamide) and teriflunomide (an inhibitor of dihydroorotate dehydrogenase, DHODH) protected 20% and 40% of mice from lethal WNV challenge, respectively. Both drugs, which are orally bioavailable and have been approved clinically for many years, may be promising therapeutics for WNV infection. Moreover, the other two DHODH inhibitors, ML390 and vidofludimus, also displayed potent activity against WNV infection in vitro and in vivo.

Preparation and separation of pure spherical cellulose nanocrystals from microcrystalline cellulose by complex enzymatic hydrolysis.

Spherical cellulose nanocrystals (CNCs), as a new and high value cellulose derivative, shows excellent application potential in many fields due to its special structure. The accurate and effective separation of pure spherical CNCs lays foundation for its further application. In this work, spherical CNCs were prepared by enzymatic hydrolysis of microcrystalline cellulose (MCC) with complex enzymes. In order to determine the optimal separation conditions of pure spherical CNCs, turbidity and Zeta potential were used to analyze the influence of pH on system stability, and the size and morphology of samples were characterized by DLS, AFM and SEM. The results showed that spherical CNCs with particle size of 24-76 nm can be separated from large particles with the help of alkali (pH = 9) dispersion and centrifugation speed of 3000 rpm. After three acid (pH = 4) washes, pure spherical CNCs were extracted and reducing sugars and enzyme proteins were removed. Compared with MCC, spherical CNCs had lower crystallinity but stronger reactivity and higher heat transfer. DTG results showed that the maximum weight loss temperature of spherical CNCs prepared by enzymatic hydrolysis was 309 °C.

Effects of Overwintering on the Survival and Vector Competence of Aedes albopictus in the Urban Life Cycle of Dengue Virus in Guangzhou, China.

The Pearl River Delta, where Aedes albopictus (Ae. albopictus) is the only vector for dengue transmission, has exhibited one of the highest dengue burdens in southern China in recent decades. However, whether dengue virus (DENV) can overwinter in Ae. albopictus in the Pearl River Delta has not been determined to date. In this study, 300 field-derived Ae. albopictus mosquitoes from Guangzhou that were infected with the predominant endemic DENV-1 strain were investigated under simulated urban balcony environment from October 16, 2016, to June 16, 2017. The vertical transmission of DENV in the infected overwintering Ae. albopictus was analyzed. The DENV infected overwintering mosquitoes were evaluated for viral load at nine-time points using reverse transcription-quantitative PCR. The vector competence of the infected overwintering Ae. albopictus was also investigated by using suckling mice. Adult mosquitoes and larvae were found during the observation period. The vertical transmission of DENV-1 was documented. The DENV-1-positive rates between overwintering males and females had no difference. The proportion of DENV-1-positive overwintering mosquitoes decreased over time and had no difference beyond three months after the experiment. Overwintering mosquitoes can spread DENV-1 to hosts. No engorged mosquitoes at an ambient temperature below 15 °C were observed. The ratio of engorged mosquitoes was positively correlated with the ambient temperature ranging from 15 to 30 °C. Our results demonstrated that DENV can overwinter in Ae. albopictus in the Pearl River Delta, Ae. albopictus is the competent vector for DENV, and maintain autochthonous dengue outbreaks in the Pearl River Delta through vertical transmission.

Preparation and application of yellow fever virus NS1 protein-specific monoclonal antibodies.

Yellow fever is an acute infectious disease that is common in Africa and South America and causes thousands of deaths annually. However, there are very few studies on yellow fever virus (YFV) antigen detection kits. As a detection target, the nonstructural protein 1 (NS1) has been successfully used in the early diagnosis of dengue virus (a member of the Flaviviridae family) infection. In this study, we used monoclonal antibody technology to prepare anti-YFV NS1 monoclonal antibodies (MAbs) and identified their immunological properties. Next, we used two mouse MAbs that can recognize different epitopes of YFV NS1 as capture and detection antibodies to establish a YFV NS1 antigen-capture enzyme-linked immunosorbent assay (ELISA). The antigen-capture ELISA displayed exclusive specificity to YFV without cross-reaction with other related members of the flavivirus family, including the dengue virus, West Nile virus, Japanese encephalitis virus. Additionally, the detection sensitivity towards the YFV culture supernatant was 103 TCID50/mL and the detection positivity rate was 95% compared with reverse transcription-polymerase chain reaction. In conclusion, this newly developed NS1 antigen-capture ELISA with high sensitivity and specificity could be used as an efficient method for the early diagnosis of YFV infection in animals or humans.

A predominant dengue virus-1 endemic strain and the vector competence of Aedes albopictus from Guangzhou City, China.

INTRODUCTION: Dengue has been a serious public health burden and dengue virus-1 (DENV-1) is the predominant strain in Guangdong province, China. Differences exist in the transmission dynamics amongAedes albopictus and DENV in different geographical regions. However, little is known about the vector competence of indigenous Aedes albopictus for the predominant dengue strain in Guangdong province, China. METHODOLOGY: In this study, the field-derivedAedes albopictus collected from Guangzhou city, Guangdong province were infected with the predominant DENV endemic strain DENV-1 GZ201401 by feeding on serially diluted artificial infectious blood or infected suckling mice. DENV-infected mosquitoes were evaluated for viral load at five-time intervals in three tissues, the head, body and legs using reverse transcription-quantitative PCR (RT-qPCR). The vertical transmission of DENV in Ades albopictus was also analysed. Suckling mice were used to assess the transmission of DENV by Aedes albopictus. RESULTS: There was no difference in infection rates between mosquitoes infected by infected suckling mice or by artificial infectious blood. The proportion of DENV-1 positive mosquitoes increased over time after an infectious blood meal, but there was no difference in the positive rate beyond 7days after the blood meal. The positive rate of DENV-1 infected mosquitoes increased with the DENV titer in the blood meal. Most of the infections the infected mosquitoes were disseminated more than 7 days after imbibing the artificial infectious blood. The median infective doses (MID50) at 7,14,21 and 28 days after artificial infectious blood meal [7, 14, 21 and 28 days post-infection (dpi)] were 7.86 × 107, 1.57 × 107, 6.39 × 106 and 4.96 × 106 TCID50 (50% tissue culture infective dose)/ml, respectively. The mosquitoes can spread DENV-1 GZ201401 to hosts as early as 3 dpi. The vertical transmission of DENV-1 was documented with a cumulative rate of 17.61%. CONCLUSION: Our results demonstrated that Aedes albopictus mosquitoes are competent vectors for DENV-1, and are capable of maintaining autochthonous dengue outbreaks in Guangdong province, China, which may have been promoted by vertical transmission.

Identification of two novel peptides with antioxidant activity and their potential in inhibiting amyloid-ß aggregation in vitro.

Two novel peptides WW4 and WW7 were evaluated for their antioxidant activity, membrane penetrance and inhibiting activity of amyloid-ß protein (Aß) aggregation. The results showed that both WW7 (10.38 ± 0.22 µmol TE per µmol) and WW4 (6.32 ± 0.77 µmol TE per µmol) possessed a significant oxygen radical absorption capacity (ORAC) and strong 1,1-diphenyl-2-picrylhydrazyl (DPPH˙) scavenging capacity (WW7, IC50 0.05 ± 0.002; WW4, 1.06 ± 0.07). Interestingly, WW7 exhibited relatively higher antioxidant activity than WW4. In addition, both WW4 and WW7 showed high cell membrane penetrance characteristics in HEK293 cells. To measure the metabolic stability of WW4 and WW7 in cells, we labelled the peptides with FITC and then analyze the co-localization with lysosomes by imaging Flow-cytometry. We found that WW7 had a lower co-localization rate (1.39%) than WW4 (8.44%), indicating that WW7 was more stable than WW4. In vivo imaging assay demonstrated that WW7 presented higher metabolic stability with a much longer stability time (2687.33 ± 54.01 min) in BALB-c nude mice than WW4 (148 ± 26.85 min), which was consistent with the in vitro result. To illustrate the potential function of antioxidant capacity, an Aß aggregation cell model was applied to examine anti-Aß aggregation ability of WW4 and WW7. Surprisingly, WW7 (23.04 ± 13.64%) had stronger anti-Aß aggregation ability but WW4 did not show obvious potential, which was due to their structure difference. The present work would offer novel insight into the activity of antioxidants and anti-Aß aggregation, and uncover the under-appreciated function of peptides in effective application in AD therapy.

Laboratory and Molecular Characterization of Dengue Viruses in a 2014 Outbreak in Guangfo Region, Southern China.

Non-specific symptoms and low viremia levels make early diagnosis of dengue virus (DENV) infection challenging. This study aimed to i) identify laboratory markers that can be used to predict a DENV-positive diagnosis and ii) perform a molecular characterization of DENVs from the 2014 Guangdong epidemic. This retrospective study analyzed 1,044 patients from the Guangdong epidemic who were clinically suspected cases of dengue. Viral RNA was detected by real-time RT-PCR, and viral-specific NS1 antigen was detected using enzyme-linked immuno sorbent assay. A molecular phylogenetic analysis was performed for the with the DENV C-prM gene junction. Patients with dengue infection had leukopenia (2.8 × 109/L), thrombocytopenia (109.0 × 109/L), elevated aspartate aminotransferase (56.0 IU/L) and alanine aminotransferase (43.5 IU/L), and prolonged activated partial thromboplastin time (APTT, 33.5 s) (all P ＜ 0.001) compared to patients without dengue. The positive predictive value of leukopenia and thrombocytopenia for DENV infection were 96.9% and 93.0%, respectively. Leukopenia, thrombocytopenia, elevated aminotransferases, and prolonged APTT were useful predictive markers for an early diagnosis of DENV infection. Phylogenetic analysis indicated that the DENVs from the 2014 epidemic were closely related to a 2010 New Delhi strain and a 2013 Guangzhou strain. The 2014 epidemic consisted of co-circulating DENV-1 genotypes I and V from multiple origins. Efficient dengue surveillance can facilitate rapid response to future outbreaks.

Lower activation-induced T-cell apoptosis is related to the pathological immune response in secondary infection with hetero-serotype dengue virus.

The available evidence suggests that dengue virus-specific T lymphocytes and cytokine storm play a pivotal role in the immunopathogenesis of plasma leakage. Investigations are underway to identify the immune profiles associated with increased or decreased risk for severe disease. In this study, CD14+ cells from the peripheral blood mononuclear cells (PBMCs) of patients who recovered from DENV-1 infection were infected with DENV-1 or DENV-2 and co-cultured with memory T cells. We found that secondary infection with DENV-2 suppresses the cell reproductive capacity but forms more cell clones and more functional cells to produce more proinflammatory factors (IFN-γ, TNF-α, IL-6, IL-8, IL-12 and IL-17) and less regulatory cytokines (IL-10, TGF-ß) which results in higher viral replication compared to secondary infection with DENV-1. Memory dengue virus-specific T cells which are induced in a primary dengue virus infection are reactivated by the heterologous serotype of dengue virus and antigen-presenting cells (APCs) during a secondary infection. Dramatically, less apoptosis and more continuous activation of T cells in secondary infection with hetero-serotype DENV were observed. This discovery which has not been reported previously may be the reasonable and vital interpretation for the cytokine storm and severe symptoms observed in secondary infection with DENV. In summary, secondary infection with hetero-serotype DENV elicits the relatively pathological immune response while secondary infection with homologous-serotype DENV induces the relatively protective immune response by activation-induced cell death (AICD) of T cells.

A survey of the 2014 dengue fever epidemic in Guangzhou, China.

In 2014, a serious dengue outbreak in Guangzhou occurred, consisting of 37 354 laboratory confirmed cases of infection. In this study, the clinical picture of dengue fever due to dengue virus (DENV) type 1 in Guangzhou was described. Clinical and laboratory data collected by studying 726 sera of suspected clinical cases from hospitals and 328 sera of healthy persons from two residence communities were analyzed during the outbreak, and 484 patients were diagnosed with an acute dengue infection. Fever, headache, congestion of the throat, and myalgia were the most typical symptoms in DENV-infected patients. Thrombocytopenia, leukopenia, and an increase in liver enzymes were significantly more common in the infected patients than in the healthy controls. Fourteen cases of silent infection were discovered among the 328 healthy persons, suggesting a DENV inapparent infection rate of 4.27% among healthy individuals. The data obtained by analyzing 212 positive sera with three methods indicated different results with different detection methods. DENV RNA should be used for early diagnoses during days 1-6 after symptom onset, immunoglobulin M (IgM) can be easily recognized after four days have passed since symptom onset and DENV isolation has a peak positive rate during days 1-3 after the onset of symptoms. A phylogenetic analysis of viral NS1 gene sequences from this outbreak indicated that the predominant isolates could be categorized as DENV-1 genotype III and had the highest homology with the India genotypes from 2009 to 2011. However, this analysis also revealed a co-epidemic of the 2013 Zhongshan and 2003 Singapore genotypes, both belonging to DENV-1 genotype I, which suggested multiple geographic origins for the 2014 epidemic of dengue 1 strains in Guangzhou.

[Research progress in the structure and function of dengue virus non-structural 1 protein].

Dengue virus (DENV) is a re-emerging disease transmitted by the Aedes mosquitoes and has become a major public health problem in southern China. Currently, no antiviral drug or effective vaccine exist to control this disease. The chimeric DENV structural protein vaccine cannot elicit balanced levels of protective immunity to each of the four viral serotypes; therefore, non-structural protein components may be required to construct an effective DENV vaccine. The Dengue virus non-structural 1 (DENV NS1) protein plays a critical role in viral pathogenesis and protective immunity. Therefore, immunity to Dengue 1-4 NS1 subtypes may be crucial for the prevention of severe disease. This review attempts to provide an overview about the structure and function of DENV NS1.

[Screening and identification of dengue virus type 2-specific antigens].

OBJECTIVE: To screen and identify dengue virus type 2 specific antigens and establish an enzyme-linked immunosorbent assay (ELISA) for detecting dengue virus type 2 antibody. METHODS: Using the bioinformatic software DNAstar and ANTHEPROT, we analyzed the hydrophilicity, flexibility, surface probability and antigenicity of dengue virus type 1-4, Japanese encephalitis virus, and Yellow fever virus M and E protein amino acid sequences, and also evaluated the influence of secondary structure. The specific epitopes of dengue virus type 2 were predicted according to the epitope location and amino acid sequence similarity, and the epitope conservation was assessed using the sequence information of different dengue virus type 2 strains in GenBank. Based on the results of bioinformatic analysis, 5 specific epitopes were amplified and inserted into the prokaryotic expression vector pET32a, which were transferred into E. coli Rosetta (DE3) for expression of the proteins. SDS-PAGE and Western blotting were used to identify the expressed proteins and test their antigenicities. The antigen selected by Western blotting was used to establish the ELISA system for dengue virus type 2 antibody detection. RESULTS: Bioinformatic analysis predicted 8 possible dengue virus type 2 specific epitopes, and 6 of them were efficiently expressed in E. coli. Western blotting confirmed 1 dengue virus type 2 specific antigen, the ELISA system for dengue virus antibody detection was successfully established using this specific antigen. CONCLUSION: We have obtained a dengue virus type 2 specific antigen and established an ELISA system for detection of dengue virus type 2 antibody.

Cloning, characterization, and biological function analysis of the SidT2 gene from Siniperca chuatsi.

The systemic RNA interference defective protein (SID)-1 plays an important role in dsRNA uptake in cells. We identified the ScSidT2 gene from the mandarin fish (Siniperca chuatsi), which is the first-studied SID-1 homolog in fish. ScSidT2 mRNA is 3380 bp long and contains a 2568-bp open reading frame that encodes an 855-amino-acid protein with an N-terminal signal peptide and ten putative transmembrane domains. Tissue distribution profile in healthy fish and expression profiles of ScSidT2 in infectious spleen and kidney necrosis virus (ISKNV)-infected fish were analyzed. Overexpression of the ScSidT2 protein in fathead minnow (FHM) epithelial cells could remarkably increase the uptake of exogenous dsRNA. In tiger frog virus (TFV)-infected FHM cells, overexpression of ScSidT2 could suppress virus production, and this mechanism could be significantly enhanced by adding virus-specific dsRNA. In mandarin fish fry cells, ISKNV infection and reproduction could be promoted when the ScSidT2 protein was neutralized with specific antisera. These results suggest that ScSidT2 could be associated with the host's antiviral defense mechanism.

[Study on E protein epitopes and primary identification of main yellow virus].

OBJECTIVE: To analysis the E protein epitopes of dengue virus type 1-4, Japanese encephalitis virus and yellow fever virus and to distinguish the shared or specific epitopes among them. METHODS: Bioinformatic software DNAStar was used to analyze the hydrophilicity, flexibility, surface probability and antigenicity of dengue virus type 1-4, Japanese encephalitis virus and yellow fever virus E protein amino acid sequences. The influence of secondary structure was also considered. Based on the bio-informatic analysis of E protein epitopes, 6 specific epitopes were amplified and inserted into prokaryotic expression vector pMAL-c2x. The vectors was then transferred into E. coli BL21 (DE3) and Rosetta (DE3). Isopropyl-beta-D-thiogalactoside (IPTG) was used to induce the expression of gene segments and SDS-PAGE were used identify the expression proteins. The antigenicity was tested, using Western blot. RESULTS: 15 shared epitopes and 47 specific epitopes were forecasted by bioinformatic analysis, and 6 specific epitopes from dengue virus type 1-4, Japanese encephalitis virus and yellow fever virus E protein were expressed in E. coli successfully. Two specific antigenic determinant from dengue virus type 1 and dengue virus type 2 were confirmed using Western blot, while the others epitopes shown no antigenic reaction property. CONCLUSION: Two specific antigenic determinant were confirmed, under Western blot.