[[Virus-like particle-based immunoglobulin M capture enzyme-linked immunosorbent assay for the detection of IgM antibodies against Chikungunya virus].

To establish a MacELISA method for the detection of IgM antibodies against Chikungunya virus (CHIKV), we prepared virus like particle (VLP) antigens of CHIKV using the whole structural protein C-E3-E2-6K-E1 encoding gene with a baculovirus expression system in Sf9 insect cells. The VLPs were purified and used to immunize Kunming mice. Then, polyclonal antibodies were purified from the samples of ascites with a protein G HiTrap SP column and labeled with horseradish peroxidase.

[Secreted expression of dengue virus type 2 envelope glycoprotein in eukaryotic cells].

Objective: To secreted express envelope glycoprotein (E) of dengue virus type 2 extracellularly.

Methods: The entire prM/E gene was amplified by RT-PCR. An optimized signal sequence gene from Japanese encephalits virus (JEV, SA14-14-2 strain) was introduced using fusion PCR.

[Identification and construction of replicon vectors of Japanese encephalitis virus (strain SA14-14-2)].

Objective: In order to lay the groundwork for studying the novel vaccine Identified.

Methods: (1) Two replicons were constructed. One's prM/E gene was deleted completely (Full AprM/E Replicon), the other's prM/E gene was deleted partially (213 bp of C terminal of E gene was reserved; Partial delta prM/E Replicon), and the deleted parts was replaced as the MCS.

[Secreted expression of dengue virus type I envelope glycoprotein in 293T cells].

Objective: To expression prM/E gene of dengue virus type I in mammalia cells.

Methods: The full-length prM/E gene of dengue virus type I strain GZ01/95 was amplified by RT-PCR, the signal peptide preceding the prM gene was added or the carboxyl-terminal 20% of DEN-1 E was replaced with the corresponding JE sequence in the meanwhile, and three of the constructions were cloned into the pcDNA5/FRT.Then they were transfected into 293T cells by lipofectamine respectively.

[Fluorescent microbeads-based multiplex detection of IgM antibodies to pathogens caused viral hemorrhagic fever].

Objective: To develop and evaluate a multiplex detection of IgM antibodies to pathogens caused viral hemorrhagic fever.

Methods: The nucleocapsid proteins (NP) of HTN, SEO, Puu MBV, Lassa, RFV and HPS viruses expressed in prokaryotic cells and purified were coupled to 7 different xMAP fluorescent microbeads. The assay was evaluated and optimized when screened against a panel of reference sera collected from HFRS patients, and compared to commonly used MacELISA Kits.

[Construcion of a chimeric Japanese encephalits virus/dengue virus-2].

The prM/E gene of DV2 was cloned into the JEV (SA14-14-2 strain) replicon vector which had been constructed previously, and the resulting recombinant plasmid was named pPartialdeltaprM/E. The constructed chimeric clone was linearized and then was transcripted into RNA in vitro. The produced RNA was transfected into BHK-21 cells.

[The differential expression of the human lung carcinoma cells infected with high pathogenic avian influenza virus A/Anhui/1/2005 (H5N1)].

Objective: To identify genes in human cells infected with high pathogenic avian influenza viruses H5N1.

Methods: The lung carcinoma cells line A549 was infected with H5N1 and H1N1, respectively. We harvested the infected cells at the different time points after infection and screened the genes with differential expression via microarray technology.

[Recombinant envelope glycoprotein domain III of dengue virus inhibit virus infection].

Objective: To observe the ability of dengue virus recombinant envelope protein domain expressed in E. coli to inhibit virus infection and induce the neutralizing antibody.

Methods: E III protein of Dengue virus serotypes 1-4 were expressed in E.

[Hantavirus mucosal vaccine through different mucosal with heat-labile enterotoxin B subunit as adjuvants].

Objective: To evaluation the effect of different mucosal vaccination pathway on hantavirus with the recombinant E. coli heat-labile enterotoxin B subunit (rLTB) as adjuvant.

Methods: The rLTB was expressed and purified.

[Generation of neutralizing recombinant human antibodies for targeting highly pathogenic avian influenza A (H5N1) virus].

Two human Fab antibodies against avian influenza A (H5N1) virus were obtained by panning a H5N1 patient-derived antibody phage library using purified virions of the H5N1 patient isolate A/Anhui/1/2005 and HA protein of the H5N1 reference viruse A/Viet Nam/1203/2004. After testing the binding properties and antiviral function to H5N1 virus, the selected Fab antibodies were converted to full human IgG antibodies with recombinant baculovirus/insect cell system. Both mAbs, AVFluIgG01 and AVFluIgG03, bound to HA in immunofluorescence assay (IFA) without cross-reaction with the other substypes of influenza A viruses (H1N1, H3N2).

[Development and application of a two-step MacELISA for the early diagnosis of hemorrhagic fever with renal syndrome].

Objective: To develop and improve a MacELISA method for the early diagnosis of hemorrhagic fever with renal syndrome (HFRS) with simplified operation procedure.

Methods: The nucleic proteins of hantavirus were labeled with horse raddish peroxidase (HRP) and used as detection antigens. A two-step MacELISA based HRP conjugated antigen was established and the detection sensitivity and specificity were compared with commonly used three-step MacELISA.

[Development and evaluation of a double antigen sandwich ELISA for the detection of total antibodies against hemorrhagic fever with renal syndrome virus].

Objectives: To develop and evaluate a method for detection of the total antibodies against hemorrhagic fever with renal syndrome (HFRS) virus with improved sensitivity and simplified operation procedure.

Methods: The nucleic proteins of hantavirus were used as coating antigens as well as detection antigens labeled with horse radish peroxidase (HRP). The operation protocol was established, optimized and compared with indirect fluorescence assay (IFA).

[Detection of IgM antibody against hantavirus by chemiluminescent enzyme-linked immunosorbent assay].

Objective: To develop a chemiluminescent enzyme-linked immunosorbent assay (CLEIA) for the detection of HTNV IgM antibody.

Methods: Black solid 96 well microplate was coated with anti-human IgM-microantibody, HRP labeled HTNV recombinant nucleotide antigen was used as detection antigen, luminol-H2O2 was used as substrate, a CLEIA was established for the detection of HFRS patient serum IgM antibody and comparison of detection sensitivity, specificity, and stability were made between CLEIA and MacELISA.

Results: Correlate coefficient of CLEIA with MacELISA is 0.

[Genotyping of the Chinese isolates of coltivirus].

Objective: To classify the Chinese isolates of Coltiviruses.

Methods: Three sets of primers were selected among them two were specific to the 9th and 12th segments of subgroup B2, and one was for the 12th segment of subgroup B1-All the Chinese isolates of Coltivirus selected in the experiment were classified according to the lengths of different amplicons of the reverse transcriptase-polymerase Chain reaction (RT-PCR). The homogenicity of the nucleic acids of the isolates BJ95-75 and YN-6 was also compared with other Coltivirus strains belonging to subgroup B2.

[Investigation of Arboviruses in Lancang river down-stream area in Yunnan province].

Objective: To investigate the epidemic state of arboviruses in the downstream area of Lancang river in Yunnan province.

Methods: Mosquitoes were collected from Lancang river downstream area (including Lancang county and Simao city) during summer in 1998 and stored in liquid nitrogen after classification. The mosquito pools were homogenized and centrifuged, then the supernatant was inoculated into C6/36 cells for virus isolation.