Evaluation of a self-prepared anti-WNV-IgG diagnostic ELISA kit with a panel of serum samples collected from the people from areas in which West Nile fever is endemic / 病毒学报

In view of that there is no report of west Nile virus infection cases in our country, evaluation the self-prepared anti-WNV-IgG diagnostic ELISA kit should be employed with the establishment of the serum sample panel collected from the entry personnel. All individuals of entry personnel were traveled from epidemic area of infectious west Nile disease. In our study, the serum samples were both detected by self-prepared anti-WNV-IgG diagnostic ELISA kit and the FDA certified kits ,which are FOCUS West Nile Virus IgG Dxselect and Panbio Dengue IgG Capture ELISA kits. The self-prepared kit and FDA certified kits were compared and assessed simultaneously. Furthermore, the specificity, repeatability and stability of the kits were also evaluated. The results indicated that no significant difference of detective rates (35. 6% for self-prepared kit vs. 32.5% for FOCUS kit, χ2 = 3. 05, P > 0.05) and good consistency (Kappa = 0.8372) between the self-prepared kit and FDA certified kits. Also, the positive coincidence rate, the negative coincidence rate and the total coincidence rate were calculated as 91.18%, 95.34% and 92.66%, respectively. The laboratory self-developed kit presented similar quality as the counterpart kits with FDA certificate. The development of our self-prepared anti-WNV-IgG diagnostic ELISA kit will provide technical support for the prevention and control of west Nile virus entry.

Removal of oral Prevotella intermedia Endotoxin by octyl phenyl polyoxyethylene ether extraction method / 中华口腔医学杂志

<p><b>OBJECTIVE</b>To investigate an effective purification method for removing endotoxin from Prevotella intermedia.</p><p><b>METHODS</b>The main protein ingredients of bacteria prepared from ammonium sulfate precipitation were further treated with octyl phenyl polyoxyethylene ether (Triton X-114), and then processed at 4°C, 37°C and 25°C. The obtained aqueous phase after at least two more cycle repeated operations was assayed for endotoxin by Western blotting, LAL-clotting method, in vitro cell stimulation and in vivo animal experiments.</p><p><b>RESULTS</b>Western blotting and LAL-clotting method demonstrated that the reduction in endotoxin level was greater than 99.99% and recovery of the proteins after endotoxin removal was greater than 90% with Triton X-114 treatment for 3 cycles. The cytokines expression level was lower in both in vitro cell stimulation and in vivo animal experiments than in untreated group (P < 0.05).</p><p><b>CONCLUSIONS</b>The extraction method provides a new choice for endotoxin removal from large volumes of the oral Prevotella intermedia.</p>

Multiplex real-time PCR method for rapid detection of Marburg virus and Ebola virus / 中华实验和临床病毒学杂志

<p><b>OBJECTIVE</b>Marburg virus and Ebola virus are acute infections with high case fatality rates. A rapid, sensitive detection method was established to detect Marburg virus and Ebola virus by multiplex real-time fluorescence quantitative PCR.</p><p><b>METHODS</b>Designing primers and Taqman probes from highly conserved sequences of Marburg virus and Ebola virus through whole genome sequences alignment, Taqman probes labeled by FAM and Texas Red, the sensitivity of the multiplex real-time quantitative PCR assay was optimized by evaluating the different concentrations of primers and Probes.</p><p><b>RESULTS</b>We have developed a real-time PCR method with the sensitivity of 30.5 copies/microl for Marburg virus positive plasmid and 28.6 copies/microl for Ebola virus positive plasmids, Japanese encephalitis virus, Yellow fever virus, Dengue virus were using to examine the specificity.</p><p><b>CONCLUSIONS</b>The Multiplex real-time PCR assays provide a sensitive, reliable and efficient method to detect Marburg virus and Ebola virus simultaneously.</p>

Expression, purification and identification of St. Louis encephalitis virus-like particles in eukaryotic cells / 中华实验和临床病毒学杂志

<p><b>OBJECTIVE</b>To express St. Louis encephalitis virus-like particles in mammalian cells, it will provide a prerequisite for further immunological diagnostic studies.</p><p><b>METHODS</b>293T-cell were transiently transfected with recombinant PreM-E plasmid. Expression and antigenicity of the purified protein were determined by transmission electron microscope (TEM), Western-Blot, immunofluorescence assay and ELISA.</p><p><b>RESULTS</b>Recombinant subviral particles, about 50 nm in diameter, were observed by TEM in the supernatant of transfected cells. The results of Western-Blot, IFA and ELISA showed the recombinant proteins retained immunoreactivity similar to those of native virus proteins.</p><p><b>CONCLUSION</b>St. Louis encephalitis virus-like particles has good antigenicity and physical appearance. It will provide a prerequisite for further immune diagnostic reagent.</p>

Transmission electron microscopy investigation of the denudation of the envelope of influenza virus treated with Nonidet P-40 and the binding influence of antibody / 中华实验和临床病毒学杂志

<p><b>OBJECTIVE</b>Through observing the morphological changes of the prepared influenza viruses (H1N1) treated with the different concentration of Nonidet P-40 solutions and added with antibody against the influenza virus using transmission electron microscopy (TEM), to explore the principle of application of the internal antibody for immunoassay of influenza virus.</p><p><b>METHODS</b>Through treating the virus samples with serial diluted Nonidet P-40 solutions from 0.01% to 0.2% and/or not adding antibody against the influenza virus, then investigating the samples by TEM to obtain the morphological changes of the virions.</p><p><b>RESULTS</b>The serial images show that the denudation degree of the virions is proportional with the rise of NP-40 concentration, and partly denuded virion image appeared at 0.1% NP-40 treatment, also under this condition no influence was observed on antibody binding to the virus.</p><p><b>CONCLUSION</b>This work demonstrated the interaction between influenza virion and its antibody under nonionic surfactants existing, which supports the advantages of sample preparation for immunoassay enveloped virus using internal antibody theoretically.</p>

Development of a universal primers PCR-coupled liquid bead array to detect biothreat bacteria / 中华预防医学杂志

<p><b>OBJECTIVE</b>To develop a fast, high-throughput screening method with suspension array technique for simultaneous detection of biothreat bacteria.</p><p><b>METHODS</b>16 S rDNA universal primers for Bacillus anthracis, Francisella tularensis, Yersinia pestis, Brucella spp.and Burkholderia pseudomallei were selected to amplify corresponding regions and the genus-specific or species-specific probes were designed. After amplification of chromosomal DNA by 16 S rDNA primers 341A and 519B, the PCR products were detected by suspension array technique. The sensitivity, specificity, reproducibility and detection power were also analyzed.</p><p><b>RESULTS</b>After PCR amplification by 16 S rDNA primers and specific probe hybridization, the target microorganisms could be identified at genus level, cross reaction was recognized in the same genus. The detection sensitivity of the assay was 1.5 pg/microl (Burkholderia pseudomallei), 20 pg/microl (Brucella spp.), 7 pg/microl (Bacillus anthracis), 0.1 pg/microl (Francisella tularensis), and 1.1 pg/microl (Yersinia pestis), respectively. The coefficient of variation for 15 test of different probes was ranged from 5.18% to 17.88%, it showed good reproducibility. The assay could correctly identify Bacillus anthracis and Yersinia pestis strains in simulated white powder samples.</p><p><b>CONCLUSION</b>The suspension array technique could be served as an opening screening method for biothreat bacteria rapid detection.</p>

Study on the morphology of influenza virus A by atomic force microscopy / 病毒学报

The aim of the study is through observing the morphology of the prepared influenza virus (H1N1) with transmission electron microscopy (TEM) and atomic force microscopy (AFM) to explore the application of AFM on the research of the external character of viruses and provide a new, simple and efficient technique for the study of the viral morphology. TEM image was obtained by negatively stained influenza virus with 1% Phosphotungstic Acid; AFM image applied the tapping mode to influenza virus without any further treatment in air at room temperature, and the morphology parameters, including length (diameter), Ra and Rq are calculated by sectional analysis. The shapes of influenza virus A are spherical, filamentous or other pleomorphous particles observed by both AFM and TEM. TEM image of influenza virus A is two-dimensional image, and viral surface has visible spikes, while AFM exhibits the three-dimensional image that can be described with several quantifiable indexes through sectional analysis. AFM phase images show viral surface clearly which is characterized by rugged feature and gear-like protuberance. As compared with TEM, AFM is a new research tool for viral morphology study with the advantages of simple sample preparing, visible interface and is intuitionistic for researchers. The surface characteristic parameters of viruses provided by AFM can be served as the main quantifiable indexes for viral morphological study.

Atomic force microscopy investigation of the denudation of the envelope of influenza virus treated with Nonidet P-40 / 中华实验和临床病毒学杂志

<p><b>OBJECTIVE</b>Through observing the morphology and topography of the prepared influenza viruses (H1N1) treated with the different Nonidet P-40 solutions using atomic force microscopy (AFM), to explore the application of AFM on the research of the internal character of viral morphology and structural virology.</p><p><b>METHODS</b>The virus samples were treated with serial diluted Nonidet P-40 solutions from 0.05% to 0.20% and then investigated by AFM with the tapping mode in air at room temperature to obtain the morphology and topography changes including height data,amplitude data and phase data for both spherical and filamentous influenza virus A.</p><p><b>RESULTS</b>The serial AFM images show that the erosion degree of the virions is proportional with the improvement of NP-40 concentration,and partly denuded virion image appeared at 0.05% NP-40 treatment, which was revealed clearly on both amplitude images and phase images.</p><p><b>CONCLUSION</b>This work demonstrated for the first time that the internal topography of influenza virion could be revealed by AFM via suitable nonionic surfactants chemical dissection.</p>