Cryosubstitution technique reveals new morphology of flavivirus-induced structures.

Cryotechniques in combination with electron microscopy were used in an attempt to obtain more precise morphological details of flavivirus-induced structures. From conventional chemical fixation procedure, proliferation of endoplasmic reticulum, formation of microtubule paracrystals and clusters of smooth membrane vesicles (with 'thread-like' enclosures) were observed. These induced changes are typical for flavivirus infections. The images obtained from cryosections were disappointing as the structures were not well preserved. On the other hand, cryosubstituted-infected cells gave revealing images of the virus-induced structures. The most obvious difference between the cryosubstituted and chemical fixed processes was on the morphology of the 'thread-like' structure. The 'thread-like' structures instead appeared as dense cores. The morphology of the virus particles was also better defined. The envelope of the virus appeared clearly differentiated from the nucleocapsid. The most important finding was that the cryosubstituted technique was able to preserve the structures of the flavivirus nucleocapsids which so far has not been convincing reported with chemical processing.