Submitted to: Microscopy and Microanalysis
Publication Type: Peer reviewed journal
Publication Acceptance Date: 1/3/2014
Publication Date: 2/20/2014
Publication URL: http://handle.nal.usda.gov/10113/60116
Citation: Kiss, G., Chen, X., Brindley, M., Campbell, P., Afonso, C.L., Ke, Z., Holl, J., Guerrero-Ferreira, R., Byrd-Leotis, L., Steel, J., Steinhauer, D., Plemper, R., Kelly, D., Spearman, P., Wright, E. 2014. Capturing enveloped viruses on affinity grids for downstream cryo-electron microscopy applications. Microscopy and Microanalysis. 20:164-174. Interpretive Summary: Newcastle disease viruses and Avian Influenza viruses circulate worldwide in chickens and in wild birds. The virulent forms of both viruses cause significant disease in poultry and the attenuated form of those viruses are very commonly isolated in wild birds. Enveloped viruses exhibit great structural heterogeneity therefore the best methods for the analysis of intact particles is are cry electron microscopy and cryo electron tomography. Here a technique of for attaching the viruses to grids will allow to better study the structure of Newcastle disease and Avian Influenza viruses using those techniques. Structural studies are fundamental for the development of antivirals that target many enveloped viruses and to understand the basis of virulence. Understanding the molecular structure of those viruses will help design better strategies for controlling disease.
Technical Abstract: Electron microscopy cryo-electron microscopy and cryo-electron tomography are essential techniques used for characterizing basic virus morphology and determining the three-dimensional structure of viruses. Enveloped viruses, which contain an outer lipoprotein coat, constitute the largest group of pathogenic viruses to humans. The purification of enveloped viruses from cell culture presents certain challenges. Specifically, the inclusion of host-membrane-derived vesicles, the complete destruction of the viruses, and the disruption of the internal architecture of individual virus particles. Here, we present a strategy for capturing enveloped viruses on affinity grids for use in both conventional electron microscopy and cryo- electron microscopy applications. We examined the utility of AG for the selective capture of human immunodeficiency virus, virus-like particles, influenza A, and measles virus. We applied nickel-nitrilotriacetic acid lipid layers in combination with molecular adaptors to selectively adhere the viruses to the AG surface. This further development of the AG method may prove essential for the gentle and selective purification of enveloped viruses directly onto EM grids for ultrastructural analyses.