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Article in Chinese | WPRIM | ID: wpr-296224


We used 293 cells to express the recombinant membrane protein of the Ebola virus. Then, the immunogenicity of the recombinant protein was studied by immunized BALB/c mice. According to the codon use frequency of humans, the gene encoding the extracellular domain of the Ebola virus membrane protein was optimized, synthesized, and inserted into the eukaryotic expression plasmid pXG-Fc to construct the human IgG Fc and Ebola GP fusion protein expression plasmid pXG-modGP-Fc. To achieve expression, the fusion protein expression vector was transfected into high-density 293 cells using transient transfection technology. The recombinant protein was purified by protein A affinity chromatography. BALB/c mice were immunized with the purified fusion protein, and serum antibody titers evaluated by an indirect enzyme-linked immunosorbent assay (ELISA). Purification and analyses of the protein revealed that the eukaryotic expression vector could express the recombinant protein GP-Fc effectively, and that the recombinant protein in the supernatant of the cell culture was present as a dimer. After immunization with the purified recombinant protein, a high titer of antigen-specific IgG could be detected in the serum of immunized mice by indirect ELISA, showing that the recombinant protein had good immunogenicity. These data suggest that we obtained a recombinant protein with good immunogenicity. Our study is the basis for development of a vaccine against the Ebola virus and for screening of monoclonal antibodies.

Animals , Antibodies, Viral , Allergy and Immunology , Ebolavirus , Genetics , Allergy and Immunology , Female , Gene Expression , Hemorrhagic Fever, Ebola , Allergy and Immunology , Virology , Humans , Immunization , Male , Mice , Mice, Inbred BALB C , Recombinant Proteins , Genetics , Allergy and Immunology , Viral Envelope Proteins , Genetics , Allergy and Immunology
Chinese Journal of Epidemiology ; (12): 368-373, 2015.
Article in Chinese | WPRIM | ID: wpr-240092


<p><b>OBJECTIVE</b>To investigate the species and distribution of mosquitoes and mosquito-borne arboviruses in Yuncheng city of Shanxi province, China.</p><p><b>METHODS</b>Mosquito samples were collected in 19 collection sites from Linyi county and Yongji city in Yuncheng city, in August, 2012. After identification and classification, all the specimens were homogenized and centrifuged to acquire supernatant before being inoculated to both C6/36 and BHK21 cells for viral isolation. Positive isolates were identified with arbovirus species-specific primers under RT-PCR, for further sequencing and phylogenetic analysis.</p><p><b>RESULTS</b>A total of 10 455 mosquitoes of 7 species in 4 genuese were collected. The predominant mosquito species in Linyi county was Culex pipens pallens (91.96%, 3 911/4 253), but the one in Yongji city was Culex tritaeniorhynchus (72.85%, 4 518/6 202). A total of 23 strains of viruses were isolated from the mosquito pools. 15 strains from Culex tritaeniorhynchus and Culex pipens pallens were identified as genotype I Japanese encephalitis virus (JEV). Four strains from Culex pipens pallens were identified as Culex flavivirus (CxFV). Three strains from Culex pipens pallens were identified as Culex pipiens pallens densovirus (CppDNV). One strain from Armigeres subalbatus and Aedes albopictus was identified as Getah virus (GETV).</p><p><b>CONCLUSION</b>Four kinds of arboviruses were isolated from the mosquito pools, including GETV and CxFV, which were isolated and documented in Shanxi province for the first time. In the city of Yuncheng, Culex tritaeniorhynchus had been the predominant species and major vector for transmitting JEV. Genotype I JEV remained the major JEV circulating in the local natural environment.</p>

Animals , Arboviruses , Genetics , China , Cities , Culicidae , Virology , Encephalitis Virus, Japanese , Genetics , Phylogeny , Species Specificity
Chinese Journal of Zoonoses ; (12): 212-215, 2015.
Article in Chinese | WPRIM | ID: wpr-460505


The Flanders virus (FLAV) is a number of family Rhabdoviridae ,contains a single‐stranded ,negative‐sense vi‐ral RNA .Here we describe a molecular detection method developed for fast measurement of FLAV based on Taqman RT‐PCR method .In this study ,FLAV specific primers and probe were designed based on the FLAV L gene sequences published in GeneBank .Quantitative standard curve of FLAV TaqMan PCR was also successfully established .The specificity and stability test showed that the system is specific and the coefficient variables were all less than 1 .7% .Quantitative standard curve based on the genomic copy was drawn ,and the lowest detectable limit (LOD) of system was 100 copies/PCR ,with higher sensitivity and stability than that of the conventional RT‐PCR assay targeting the same gene .

Protein & Cell ; (12): 901-903, 2013.
Article in English | WPRIM | ID: wpr-757540


Rabies is an acute, progressive encephalitis caused by infection with rabies virus (RABV). It is one of the most important zoonotic infections and causes more than 70,000 human deaths annually ( ). It has long been held that a rabies infection is lethal in humans once the causative RABV reaches the central nervous system (CNS); however, this concept was challenged by the recent recovery of a small number of rabies patients. An analysis of these patients revealed that the bloodbrain barrier (BBB) played a major role in protection against the virus. The main reason for the survival of these patients was enhanced BBB permeability after infection with the causative agent (usually bat-originated RABV showing reduced pathogenicity), which allowed immune cells to enter the tissues of the CNS and clear the infection (Willoughby et al., 2005). These findings have been confirmed in animal infection experiments (Wang et al., 2005; Roy and Hooper, 2007, 2008; Faber et al., 2009). Thus, the BBB has attracted the attention of scientists interested in the pathogenesis of, and therapeutic approaches, for rabies. This paper introduces the role of the BBB in rabies infections and protection of the CNS and provides insight into future treatments for patients with clinical rabies.

Animals , Blood-Brain Barrier , Allergy and Immunology , Physiology , Virology , Disease Reservoirs , Humans , Rabies , Metabolism , Virology , Rabies virus , Virulence , Physiology
Article in Chinese | WPRIM | ID: wpr-250548


<p><b>OBJECTIVE</b>To prepare purified and concentrated coltivirus high titer antigen in order to further detect antibodies against coltivirus in serum sample of patients.</p><p><b>METHODS</b>The coltivirus in C6/36 cells was cultured and harvested at different time, and the titer was titrated. The virus was purified and concentrated by polyethylene glycol (PEG), and stored at -20 degrees and 4 degrees, with and without glycerol, respectively, then the titer of coltivirus antigen was tested by indirect ELISA. By using the antigen, coltivirus antibodies in serum samples from both suspected Japanese encephalitis (JE) and viral encephalitis (VE) patients were detected.</p><p><b>RESULTS</b>The highest titer of coltivirus was found at 3-4 weeks of culturing. The antigen titer could be maintained at least for 6 months, especially antigen with glycerol either at 4 degrees or at -30 degrees even for two years. Totally 1141 serum samples from patients diagnosed clinically as JE and VE were tested. The results showed that 130 samples were coltivirus IgM antibody positive and the average positive rate was 11.4% (130/1141). Among 41 samples of paired-serum from patients in Guangzhou Children's Hospital, 9 samples were positive, the positive rate was 22.0% (9/41) in which 5 samples were diagnosed clinically as VE.</p><p><b>CONCLUSIONS</b>Stable and purified coltivirus antigen was obtained in order to test coltivirus antibodies as well as development of kits. Coltivirus probably can cause summer-autumn encephalitis in China.</p>

Antibodies, Viral , Blood , Antigens, Viral , Cell Line , Coltivirus , Allergy and Immunology , Cryopreservation , Methods , Enzyme-Linked Immunosorbent Assay , Humans , Reoviridae Infections , Blood