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Dengue virus (DENV) is the most widely transmitted arbovirus in the world. Due to the lack of diagnostic technology to quickly identify the virus serotypes in patients, severe dengue hemorrhagic fever cases caused by repeated infections remain high. To realize the rapid differential diagnosis of different serotypes of DENV infection by immunological methods, in this study, four DENV serotype NS1 proteins were expressed and purified in mammalian cells. Monoclonal antibodies (MAbs) against NS1 protein were obtained by hybridoma technology after immunizing BALB/c mice. Enzyme-linked immunosorbent assay, indirect immunofluorescence assay, dot blotting, and Western blotting were used to confirm the reactivity of MAbs to viral native NS1 and recombinant NS1 protein. These MAbs include not only the universal antibodies that recognize all DENV 1-4 serotype NS1, but also serotype-specific antibodies against DENV-1, DENV-2 and DENV-4. Double antibody sandwich ELISA was established based on these antibodies, which can be used to achieve rapid differential diagnosis of serotypes of DENV infection. Preparation of DENV serotype-specific MAbs and establishment of an ELISA technology for identifying DENV serotypes has laid the foundation for the rapid diagnosis of DENV clinical infection.

The N-terminal segment (FR-1) of the heavy chain (VH) of antibodies may have a great impact on IgG secretion in Escherichia coli and other hosts. Decrease in secretion may be caused by a single amino acid change in the framework region. To investigate the high antibody expression in mammalian cells, we designed the site-directed mutagenesis of the FR-I of the pCMV-RV/VH gene,which expressed the immunoglobulin heavy chain of human anti-Rabies virus antibody. Mutating Glu (H6) to Gln could improve both antibody secretion and affinity. The immunofluorescence assay indicated that both the secretion-deficient antibodies and the secretion- efficient antibodies could be transcribed and translated intracellularly, and led into ER,then transferred to Golgi apparatus,and the difference in secretion may relate to the contribution of the FR-I to the folding and assembly of the antibody. In this study, we have confirmed experimentally that the nature of residues H6 in antibody heavy chains indeed determines the antibody secretion in mammalian cells. These results also provide the basis for antibody production.

Previously, an mAb 10F7 was developed against H5N1 hemagglutinin, which was highly specific to 34 different H5N1 strains and showed good neutralizing activity. In the present study, the single-chain fragment of the antibody was cloned into a prokaryotic vector and then expressed in E. coli. The activity of the scFv was tested in hemagglution inhibition and neutralization experiment. Two H5N1 virus strains were inhibited to bind erythrocyte cells by the scFv while the H9 virus was not. Also, five H5N1 virus strains were neutralized during infecting MDCK cells. These results showed an approachable method for developing therapeutic antibody to H5N1 virus.

Objective: To explore the possibility of using an endovascular microcoil as a gene delivery system., Methods: Anti-adenoviral monoclonal antibodies were covalently attached to the collagen-coated surface of platinum microcoil. These antibodies were used to tether adenovirus encoding green fluorescent protein (Ad-GFP). Cell culture studies with rat arterial smooth muscle cells (A10) assessed transduction on or near the coil. Platinum coils coated with Ad-GFP were implanted into the ligated common carotid artery (CCA) of adult rats in a model of arterial stasis and pressurization. After 7 days, CCA segments were harvested, and coils were removed for histopathology and GFP expression studies, while organs were evaluated by polymerase chain reaction to assess viral biodistribution., Results: In cell culture studies, GFP-positive smooth muscle cells were detected only on the platinum coil surface. After 7 days, GFP was detected on the harvested platinum coil and in the organizing thrombus within the CCA according to fluorescence microscopy and immunohistochemistry. Morphometric analyses revealed that (13.3 +/- 2.0)% of cells within the organized thrombus were transduced with Ad-GFP via the gene delivery system. Ad-GFP was not detectable by polymerase chain reaction in lung, liver, or kidney., Conclusions: Gene delivery endovascular microcoils represents an interventional device-based gene therapy system that can serve as a suitable platform for either single or multiple gene therapy vectors.

Objective: To investigate the epidemiological significance of hemorrhagic fever with renal syndrome virus (HFRSV) infection in domestic pigs in Shandong province, and study the role of domestic pigs in the prevalence of HFRS., Methods: Epidemiological investigation was performed in 4 cities of Shandong province. Reversed passive hemagglutination assay (RPHA), reversed passive hemagglutination inhibition (RPHI), HPR-SPA, immunofluorescent antibody (IFA), and reverse transcriptional PCR (RT-PCR) were used to detect antigen and antibody of HFRSV., Results: HFRSV antigen and antibody were detected in the heart, liver, lung, spleen, kidney, blood, urine, and stool of domestic pigs as well as in the sewage of the pigpen facilities. The positivity rate of HFRSV antigen ranged from 3.33% to 5.00% in the organ of pigs, and HFRSV positivity rate in the blood, urine, stool, and sewage was 3.67%, 7.04%, 2.51%, and 5.56%, respectively, with a total serum antibody positivity rate of 1.96%. The virus was isolated from the HFRSV antigen-positive samples, and could infect many organs after artificial infection of the pigs. HFRSV antigen can be detected in suckling rat brain and histopathological examination suggested transient pathological changes in such organs as the liver, lung and kidney. HFRSV may proliferate in pigs and was discharged through multiple routes., Conclusion: Domestic pigs can be the host of HFRSV for the viral transmission.

Aim: To obtain mimic epitopes of monoclonal antibody (mAb) against Hantaan virus, and identify preliminarily their immunological characteristics., Methods: Group-specific mAbs F3 and B11 were used as selective molecules for biopanning. After biopanning, the positive phages were identified by ELISA and DNA sequencing. Among the positive phages, F3-8, B11-18, B11-20 and B11-24 were selected to immunize BALB/c mice three times, respectively. The antiserum was then titered and confirmed by sandwich ELISA and competition ELISA., Results: After 3 to 4 rounds of effective screening, the majority of the selected clones were found able to react to mAb F3 or B11 in a dose-dependent manner, but not to BSA, rHBsAg or other unrelated mAbs. The amino acid sequences of the clones binding to mAb F3 contained an identical sequence MHGPTKNQMWHT, which had higher homology to 750-759 amino acids between a pair of cysteines within glycoprotein G2 of HTNV/SEOV, while those of clones binding to mAb B11 had no evident homologous regions within HTNV/SEOV proteins. The specific antibodies increased significantly following immunization with phage peptides, which indicated that the phage-displayed peptides had not only good antigenicity, but also strong immunoreactivity., Conclusion: The phage-displayed peptides could mimic the epitope of HFRSV antigen, which would provide the potential for preparing more effective epitope-based vaccines and specific diagnostic reagents.

Observational and experimental studies on the relationship between domestic pigs and epidemic hemorrhagic fever (EHF) were carried out during 1986 to 1994 with reverse passive hemagglutination (RPHA), reverse passive hemagglutination inhibition (RPHI), horse-radish peroxidase-staphylococcal protein A (HRP-SPA), indirect immunofluorescence assay (IFA) and reverse transcript polymerase chain reaction (RT-PCR) to understand the roles of domestic pigs played in the epidemic areas. Results showed domestic pigs could infect epidemic hemorrhagic fever virus (EHFV) naturally or experimentally, and EHFV could disseminate to many organs of the animals and caused transient pathologic changes in them. EHFV could duplicate within the animal bodies and be excreted out of the bodies with their excreta. Proportion of inapparent infection of EHF in people closely exposed to domestic pigs was significantly higher than in those without it. It indicated domestic pigs maybe played an important role in EHF as a reservoir host.

In this experiment, we established a rat model of EHFV infected by abdominal cavity, and observed the effect of moxibustion on the changes of antigens, antibodies, neural transmitters in blood and tissue on 60 weanling Wistar rats with EHF. The rats were divided into three groups: normal control group (N. group), EHF control group (C. group) and EHF with moxibustion group (M. group) at random. Moxibustion was given at "Shenshu" for 30 minutes daily, 7 days. The 14th day after infection of EHFV, blood and lungs of the rats were fetched. The results obtained are as follows: 1. The effect value of EHF antibodies dilution: in C. group, it is 1:267 +/- 82 (n = 8), in M.group, it is 1:336 +/- 176 (n = 10), there is a significantly different of specific antibodies between C.group and M.group. The results show that moxibustion had an effect of enhancing the function of immune system in rats with EHF. 2. The examination rate of EHFV antigens in lungs, in C.group, it is 62.5% (n = 8), in M.group, it is 49.4% (n = 16). The results show that moxibustion may enhance the immune function and the effect of eliminating virus of lymphocytes in lungs. 3. The value of BUN in serum: in N.group, it is 15.3 +/- 2.50 (n = 10), in C.group, it is 24.63 +/- 7.24 (n = 10), and in M.group, it is 16.36 +/- 4.59 (n = 26). There is a significantly different between C.group and N.group or M.group (P less than 0.001). There is no significantly different between N.group and M.group (P greater than 0.05).(ABSTRACT TRUNCATED AT 250 WORDS)