Adaptor-ultracentrifugation to improve the sensitivity of virus detection using electron microscopy

Direct ultracentrifugation of virus particles to specimen grid is one of the important techniques for diagnostic electron microscopy(EM).In this study,we attempt to establish a method of adaptor-ultracentrifugation to achieve the same aim of enriching viruses directly on the EM grid.Adaptor with a flat-bottomed cavity was designed and produced,which has an ultracentrifugation tubelike shape.Formvar-coated grid can be loaded on the cavity bottom.Virus-containing samples are added to the cavities,then the adaptors can be installed on the swing rotor as ultracentrifugation tubes.After ultracentrifugation,grids are picked out and subjected to negative staining,followed by EM examination.Recombinant adenovirus(Ad) and recombinant adenovirus-associated virus(AAV) were chosen as the models to compare the routine droplet method with the adaptorultracentrifugation.The results showed that the adaptor-ultracentrifugation method were about 50 to 200 times more sensitive than the droplet procedure.The similar effectiveness was also observed when the adapter-ultracentrifugation was used to detect the human diarrhea Ad(Ad41) cultivated in cells.If adapter-ultracentrifugation was combined with the immune clumping method to prepare EM specimen,the sensitivity could increase about 1 to 2 magnitudes compared to the conventional immune clumping.These data demonstrated that adapter-ultracentrifugation can enrich virus particles very effectively and can be utilized to detect viruses in diagnostic EM.