ABSTRACT
Objective@#To understand the viral etiology of a clustered case of human infection outbreak in the middle school of Huai’an city.@*Methods@#Nasopharyngeal swab samples from patients were collected and rapidly detected by Real-time RT-PCR and the target virus isolated in cells. Furthermore, HA1 segments of target virus were amplified by RT-PCR and sequenced. The genetic and phylogenetic analysis based on HA1 genes was computed.@*Results@#Influenza A(H1N1)pdm09 viral nucleic acid in 11 nasopharyngeal swab samples from patients in the outbreak were positive. Compared to the vaccine strains A/California/07/2009, the Huai’an isolates, nucleotide identity was 97.7%-98.1%, and amino acid identity was 96.6%-97.4%. Phylogenetic analysis of HA1 segment sequences indicated that the Huai’an strains from the outbreak were related closely to the viruses isolated in the year of 2014. Sequence analysis indicated that the Huai’an isolates had no amino acid substitution in the receptor binding sites and glycosylation sites, while in the Ca1 of antigenic determinant of HA1 the Huai’an isolates had an amino acid substitution of S for T at 220.@*Conclusions@#The pathogen of the clustered case of human infection was Influenza A(H1N1)pdm09 virus. Though the Huai’an isolates had one animo acid substitution in the Ca1 of antigenic determinant, the antigenicity characteristic remained unchanged.
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Objective To establish a novel method by integrating immunomagnetic bead enrich-ment with immunochromatography for the detection of influenza A virus. Methods The immunomagnetic beads were prepared by using EDC/NHS method and then coupled with monoclonal antibodies against influ-enza A virus. A direct immunomagnetic beads-based immunochromatography for the detection of influenza A virus was developed by using double-antibody sandwich method and immunochromatography, which was fur-ther combined with immunomagnetic separation to establish the novel integrated method of immunomagnectic bead enrichment and immunochromatography. Clinical throat swab samples collected from patients with influ-enza A virus infection and healthy subjects were analyzed by the novel method and the results were compared with those by using the conventional colloidal gold immunochromatography to evaluate the specificity, sensi-tivity and positive coincidence rate of this established method. Results The direct immunomagnetic beads-based immunochromatography and the colloidal gold immunochromatography showed no significant differences in specificity and sensitivity and could be used to identify influenza A virus-positive samples with cycle threshold ( Ct) values less than or equal to 22 obtained by real-time PCR assay. The integrated method could identify positive samples with Ct values less than or equal to 28, indicating that the novel method was more sensitive. Conclusion The novel method by integrating immunomagnetic bead enrichment with immunochroma-tography was successfully established and suitable for the rapid and on-site detection of influenza A virus.
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Objective To explore the application of atomic force microscopy( AFM ) on the research of morphology of the rabies viruses. Methods To prepare the rabies virus CTN-1v strains by ultracentrifugation, and observe it with transmission electron microscopy (TEM) which negatively stained by phosphotungstic acid. Then study the morphology of rabies virus with AFM based on the result of TEM. AFM image applies the tapping mode to rabies virus without any further treatment in air at room temperature. Results The TEM image is two-dimensional image which can be seen the classical bullet-shaped structure,and the spike structure can also be seen. The AFM image showed the rabies virus morphology with three-dimensional image which can shows the characteristics of the virus surface and edge. The rabies virus particle was successfully observed by TEM or AFM methods. Conclusion It's the first time to get the three-dimensional morphological structure of rabies virus by atomic force microscopy, compared with transmission electron microscopy, AFM is a new research tool for viral morphology study with the advantages of simple sample preparing and intuitionistic and visible interface for researchers.
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Objective To observe and compare the atomic force microscopy (AFM) images of Yersinia pestis EV76 and the changes in the morphology of the bacteria treated with normal serum and F1 antibody from rabbit,and to explore the immunoassay method to detect Yersinia pestis by AFM. Methods The Yersinia pestis were treated with normal serum and F1 antibody from rabbit and control buffer. All the prepared samples were observed and analyzed by AFM. The changes in the cell surface structures were probed and characterized through sectional analysis,especially the changes of Ra and Rq value. Results The normal morphology of Yersinia pestis was oval in shape with a relatively smooth surface, the size dimension of which was about 1.1-1.3 μm in length with a section profile of 0.8-1.0 μm in width and 0.04-0.06 μm in step height. The step height of the bacteria treated with the normal serum and F1 antibody was obviously enlarged. The shape of the bacteria treated with F1 antibody changed irregularly. Furthermore, the surface of the bacteria was more roughened. Conclusion The morphological characters of Yersinia pestis has been acquired through its AFM images. The morphology of Yersinia pestis treated with F1 antibody has changed greatly, and the index of roughness can be regarded as the distinguished index to detect Yersinia pestis by AFM.