Virus-like particle-based immunoglobulin M capture enzyme-linked immunosorbent assay for the detection of IgM antibodies against Chikungunya virus / 病毒学报

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. A MacELISA method for the detection of IgM antibodies against CHIKV was assembled with goat anti-human IgM antibody, VLP antigens and an enzyme-labeled polyclonal antibody. The results were evaluated with a serum panel containing serum samples from laboratory-confirmed CHIK, HFRS patients, healthy donors, and commercially available CHIKV IgM as a quality control. It was shown that the MacELISA had a specificity of 99% (99/100), the coefficients of variation (CoV) within a plate were <10%, and the CoV of different ELISA plates in terms of the plate variation coefficient was <15%. A comparative analysis was performed to compare the current method against a commercial CHIKV IgM antibody detection kit for IIFA-IgM. The detection limit of MacELISA was significantly lower than that of the IIFA-IgM commercial kit (P< 0.0001). Here, we demonstrate that the VLP-based MacELISA is a promising tool for the early diagnosis and epidemiological investigation of CHIKV infection, with a high level of sensitivity and specificity for the detection of IgM antibodies against CHIKV.

Secreted expression of dengue virus type 2 envelope glycoprotein in eukaryotic cells / 中华实验和临床病毒学杂志

<p><b>OBJECTIVE</b>To secreted express envelope glycoprotein (E) of dengue virus type 2 extracellularly.</p><p><b>METHODS</b>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. The impact of E protein transmembrane and cytoplasmatic domains was compared by amplifying prM and E with full length of E gene, with 20% truncation of the E gene at 3' terminus and one chimeric gene, which was generated by replacing the 3' terminal 20% region of E gene with the corresponding sequence of JEV (SA14-14-2 strain). The PCR segments were inserted into the NheI and NotI sites of pcDNA5/FRT vector or into the NheI and XhoI sites of pAcUW51-M. Then they were transfected into 293T cells or Sf9 cells respectively. The expression and secretion of E protein were detected by immunofluorescence assay (IFA) and Western Blot.</p><p><b>RESULTS</b>After transected into 293T cells or Sf9 cells, all constructs expressed E protein intracellularly indentified by IFA while only two plasmids could secret detectable E protein into tissue culture using Western Blot analysis.</p><p><b>CONCLUSION</b>Signal peptide as well as the transmembrane and cytoplasmatic domains is crucial for the secretion of dengue E protein.</p>

Construcion of a chimeric Japanese encephalits virus/dengue virus-2 / 病毒学报

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. Five to seven days later, CPE could be observed on the transfected BHK-21cells, and then the supernatant containing the chimeric virus was collected. The Supernatant was inoculated to BHK-1 cells and C6/36 cells, respectively. CPE could be observed about 4 days post the infection of C6/36cell with the chimeric virus. The results from RT-PCR, IFA, Western blot showed that the virus contained the chimeric RNA and the envelop protein of DV2. However, the chimeric virus could not be passaged in BHK-21 cell. The successful construction of the infectious clone JE/DEN-2 laid the basis for the further research of the DV vaccine.

Secreted expression of dengue virus type I envelope glycoprotein in 293T cells / 中华实验和临床病毒学杂志

<p><b>OBJECTIVE</b>To expression prM/E gene of dengue virus type I in mammalia cells.</p><p><b>METHODS</b>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. The expression of recombinant proteins were identified by indirect immuno-fluorescence assay(IFA) as well as Western blot.</p><p><b>RESULTS</b>In the cytoplasm of 293T cells transfected with all the recombinant plasmids DNA, the expressed products for gene of dengue virus type I were confirmed by IFA. The secreted expression products for gene of dengue virus type I specific protein bands were confirmed by Western blot only existing in the cell supernatants transfected with the modified recombinant plasmids DNA.</p><p><b>CONCLUSION</b>The prM/E protein of dengue virus type 1 were expressed in 293T cells transfected with all the three recombinant plasmids DNA. The prM/E protein was obtained secretion after transfecting the modified recombinant plasmids adding a signal peptide preceding the prM gene or replacing the carboxyl-terminal 20% of E with the corresponding JE sequence.</p>

Identification and construction of replicon vectors of Japanese encephalitis virus (strain SA14-14-2) / 中华实验和临床病毒学杂志

<p><b>OBJECTIVE</b>In order to lay the groundwork for studying the novel vaccine Identified.</p><p><b>METHODS</b>(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. (2) Replicons RNA were which will use the JEV as the vector, replicon vectors of JEV was constructed and transfected into BHK-21 cell. After 24, 48, 72, 96 h, method of real-time PCR was used to identify Replicons' replication ability. (3) YFP gene was inserted into the MCS of those two replicons. Their RNA was transfected into BHK-21 cell. Expression of YFP was tested by the fluorescence microscopy and flow cytometer.</p><p><b>RESULTS</b>(1) After the two replicons RNA were transfected into BHK-21 cell, as time went by, the quantity of RNA increased. (2) After RNA of the replicons with YFP were transfected into BHK-21 cell, increasing trend of fluorescent signal and rate of YFP positive cell was observed and tested.</p><p><b>CONCLUSION</b>Full delta prM/E Replicon and Partial delta prM/E Replicon have the ability to duplicate itself and express the foreign protein.</p>

Fluorescent microbeads-based multiplex detection of IgM antibodies to pathogens caused viral hemorrhagic fever / 中华实验和临床病毒学杂志

<p><b>OBJECTIVE</b>To develop and evaluate a multiplex detection of IgM antibodies to pathogens caused viral hemorrhagic fever.</p><p><b>METHODS</b>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.</p><p><b>RESULTS</b>For detection of anti-NP antibodies, the sensitivity and specificity of the assay were comparable to a commonly used MacELISA kit, but it could detect different antigen specific antibodies in one reaction simultaneously.</p><p><b>CONCLUSION</b>A robust, rapid and multiplex assay based on NPs could be developed via Luminex xMAP platform for laboratory diagnosis of viral hemorrhagic fever and seroepidemiological investigation.</p>

Generation of neutralizing recombinant human antibodies for targeting highly pathogenic avian influenza A (H5N1) virus / 病毒学报

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). The cross-reactivity of the two antibodies for different strains of H5N1 was tested in vitro by micro-neutralization assays. In vitro, mAb AVFluIgG01 potently neutralized not only the selected well-characterized Clade 2 H5N1 viruses isolated from mainland of China except A/Guangdong/1/2006, but also the Clade 1 representative isolate A/Viet Nam/1203/2004; and AVFluIgG03 neutralized all the selected Clade 2 H5N1 viruses isolated from mainland of China, but had no neutralizing activity with the Clade 1 H5N1 virus A/Viet Nam/1203/2004. The results bring new prospect for the prophylaxis or treatment of H5N1 virus infection and may provide a clue for novel vaccine development.

Development and application of a two-step MacELISA for the early diagnosis of hemorrhagic fever with renal syndrome / 中华实验和临床病毒学杂志

<p><b>OBJECTIVE</b>To develop and improve a MacELISA method for the early diagnosis of hemorrhagic fever with renal syndrome (HFRS) with simplified operation procedure.</p><p><b>METHODS</b>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.</p><p><b>RESULTS</b>This method could be used to detect hantanvirus specific IgM with high sensitivity and specificity from human patient serum. There was not significant difference from commonly used three-step MacELISA and the sensitivity and specificity were 100%.</p><p><b>CONCLUSION</b>This method is simple, sensitive and rapid in operation, and therefore could be used for the early diagnosis of HFRS.</p>

Hantavirus mucosal vaccine through different mucosal with heat-labile enterotoxin B subunit as adjuvants / 中华实验和临床病毒学杂志

<p><b>OBJECTIVE</b>To evaluation the effect of different mucosal vaccination pathway on hantavirus with the recombinant E. coli heat-labile enterotoxin B subunit (rLTB) as adjuvant.</p><p><b>METHODS</b>The rLTB was expressed and purified. Take the inactivated hantavirus strain 84Fli as vaccine, and immunized C57 BL/6 mice through intranasal, oral and vaginal respectively. Specific IgG and sectory IgA were detected by ELISA in serum, and vaginal washing samples respectively.</p><p><b>RESULTS</b>The rLTB was efficiently expressed under the induction of lactose, identified by western blotting and GM-1 binding experiment. The vaccination through intranasal, oral and vaginal, can induce IgG and sectory IgA response.</p><p><b>CONCLUSION</b>Inactivated hantavirus can produce mucosal immune response with rLTB as adjuvants through intranasal, oral and vaginal vaccination respectively.</p>

Recombinant envelope glycoprotein domain III of dengue virus inhibit virus infection / 中华实验和临床病毒学杂志

<p><b>OBJECTIVE</b>To observe the ability of dengue virus recombinant envelope protein domain expressed in E. coli to inhibit virus infection and induce the neutralizing antibody.</p><p><b>METHODS</b>E III protein of Dengue virus serotypes 1-4 were expressed in E. coli BL21(DE3) then purified. Recombinant proteins were tested to inhibit DV2 from infecting BHK-21 cell. Rabbits were immunized with recombinant proteins to produce anti-E III serum. Antibody titers were determined by neutralizing assay.</p><p><b>RESULTS</b>The recombinant E III proteins of Dengue virus serotypes 1-4 were expressed in E. coli. They effectively protected BHK cells in culture against DV2 infection. All four type anti-E III sera can neutralize DV2 but their efficacies are different.</p><p><b>CONCLUSION</b>proteins of dengue virus expressed in E. coli can directly inhibit DV2 infection. Neutralizing antibodies were induced by E III proteins. Both E III protein of dengue virus and the neutralizing antibodies they induced are more efficient in inhibiting homologous dengue serotypes infection than heterologous serotypes.</p>

The differential expression of the human lung carcinoma cells infected with high pathogenic avian influenza virus A/Anhui/1/2005 (H5N1) / 中华实验和临床病毒学杂志

<p><b>OBJECTIVE</b>To identify genes in human cells infected with high pathogenic avian influenza viruses H5N1.</p><p><b>METHODS</b>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. The candidate genes were selected and confirmed by quantitative real-time PCR.</p><p><b>RESULTS</b>The spectrum of genes with the differential expression in the cells infected with H5N1 was obtained and 16 candidate genes were identified in the cellular apoptosis pathway, mTOR pathway, and the cellular immunity as well.</p><p><b>CONCLUSIONS</b>Our results suggest that H5N1 exert a stronger impact on eliciting apoptosis of infected cells than the common influenza virus H1N1.</p>

Development and evaluation of a double antigen sandwich ELISA for the detection of total antibodies against hemorrhagic fever with renal syndrome virus / 中华实验和临床病毒学杂志

<p><b>OBJECTIVES</b>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.</p><p><b>METHODS</b>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).</p><p><b>RESULTS</b>The specificity of this method was 100 percent in the test of different human sera and 4-8 times more sensitive than IFA. And, it is simpler without requiring any change of the reagents, different sources of samples did not affect the results of the test.</p><p><b>CONCLUSION</b>This method is specific, sensitive and simple for detection of the total antibodies in sera against hantavirus, could be used for the screening of Hantavirus infection in human and host rodent animals.</p>

Detection of IgM antibody against hantavirus by chemiluminescent enzyme-linked immunosorbent assay / 中华实验和临床病毒学杂志

<p><b>OBJECTIVE</b>To develop a chemiluminescent enzyme-linked immunosorbent assay (CLEIA) for the detection of HTNV IgM antibody.</p><p><b>METHODS</b>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.</p><p><b>RESULTS</b>Correlate coefficient of CLEIA with MacELISA is 0.97; detection sensitivity of CLEIA is 100 percent while that of MacELISA is 92.1 percent; detection specificity of CLEIA and MacELISA are both 100 percent; coefficient of variance for intra-assay and inter-assay of CLEIA are both less than 15 percent, which are comparative with MacELISA.</p><p><b>CONCLUSION</b>The established method of CLEIA is a sensitive, selective, and stable method; it is suitable for the early detection of HFRS patient serum IgM antibody.</p>

Investigation of Arboviruses in Lancang river down-stream area in Yunnan province / 中华实验和临床病毒学杂志

<p><b>OBJECTIVE</b>To investigate the epidemic state of arboviruses in the downstream area of Lancang river in Yunnan province.</p><p><b>METHODS</b>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. New isolates were identified by neutralization test(NT), ELISA, immunofluorescence assay(IFA) and polyacrylamid gel electrophoresis(PAGE).</p><p><b>RESULTS</b>Totally 22 isolates of arbovirus were obtained from 233 mosquito pools by inoculation of C6/36 cells and positive rate of the isolation was 9.4%. Ten strains were resistant to both ether and 5 prime-IDU. So they were non-enveloped double-stranded (ds) RNA virus. Twelve segmented RNAs were shown by PAGE and PAGE profiles from the ten strains were 6-6 with minor variation. The isolates can be neutralized by immunized mouse ascites fluid of BJ95-75 strains of coltivirus by NT, and reacted with monoclonal antibody against BJ95-75 by ELISA. These viruses were identified as coltivirus. Nine isolates were sensitive to ether and resistant to 5 prime-IDU. So they were non-enveloped RNA viruses. PAGE showed 10 segmented RNA, and they were identified to be orbiviruses. Three isolations were sensitive to ether. One of them can be neutralized with JEV A2 strain antibody by NT and was positive to the homologous antibody by IFA. It was identified being strain of JE virus. One strain(YN92-4) can be reacted with anti-bunyavirus group specific immune ascites fluid by both IFA and ElISA, but reacted neither with anti-alpha virus group, nor with anti-flavivirus group JE virus ascites fluid. The virions are spherical and about 87 nm in diameter with surface projections by negative staining. Conclusion Twenty-two isolates have been obtained from wild caught-mosquitoes of Lancang river down-stream area in Yunnan province. Among them ten, nine, one and one were identified as coltivirus, orbivirus, JE virus and bunyavirus, respectively. One is under identification. This is the first report on bunyavirus isolated from mosquitoes in China.</p>

Genotyping of the Chinese isolates of coltivirus / 中华实验和临床病毒学杂志

<p><b>OBJECTIVE</b>To classify the Chinese isolates of Coltiviruses.</p><p><b>METHODS</b>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.</p><p><b>RESULTS</b>With the primers 12-854-S/12-B2-R, which were specific to the 12th segment of Coltivirus subgroup B2-850 bp amplicons were obtained from Beijing isolate BJ95-75 and all the Yunnan isolates such as YN-6, -67-1, -68-1, -69, -70-1, -70-2, -90, -92-2, -93 of Coltivirus 492 bp DNA fragments were also amplified from all of them with the segment 9th specific primers 9-JKT-S/9-JKT-R. However no positive results were obtained from Northeast isolates NE97-12, NE97-31 and control viruses YN-99(Orbivirus),YN-151-1(JEV) with the same two sets of primers. With 12-B1-S/12-B1R primers specific to the 12th segment of subgroup B1, no amplicons of right length were obtained from any of the Chinese isolates of Coltivirus and the control viruses. When compared the nucleic acid sequences of BJ95-75 and YN-6 with other Coltivirus strains such as Bannavirus, JKT6423, JKT6969, JKT7043, the amplicons from segment 12th of these two strains had more than 89.4% homology with the other strains, especially to the earlier Chinese isolate Bannavirus, the homolog was more then 98.9%. Nearly 96.5% and 99.2% of the nucleic acids of the amplicons from segment 9th of the two strains were being homologous to Bannavirus and about 84.0% to JKT6423, which had been classified into type B2a. But the maximal homogenicity was about 53% when compared with the other two coltivirus strains. JKT6969 and JKT7043 which had been classified into type B2b.</p><p><b>CONCLUSION</b>Genotyping the recent Chinese isolates of coltivirus for the first time in our country. Most of the Chinese isolates belong to subgroup B2, more exactly type B2a. The Northeast isolates NE97-12 and NE97-31 were not correctly grouped with the available primers.</p>