Imaging Bunyavirus Infections by Transmission Electron Microscopy: Conventional Sample Preparation vs High-Pressure Freezing and Freeze-Substitution.

Transmission electron microscopy significantly contributed to unveil the course of virus entry, replication, morphogenesis, and egress. For these studies, the most widely used approach is imaging ultrathin sections of virus-infected cells embedded in a plastic resin that is transparent to electrons. Before infiltration in a resin, cells must be processed to stabilize their components under the observation conditions in an electron microscope, such as high vacuum and irradiation with electrons. For conventional sample preparation, chemical fixation and dehydration are followed by infiltration in the resin and polymerization to produce a hard block that can be sectioned with an ultramicrotome. Another method that provides a superior preservation of cell components is high-pressure freezing (HPF) followed by freeze substitution (FS) before resin infiltration and polymerization. This chapter describes both procedures with cells infected with Bunyamwera virus (BUNV), a well characterized member of the Bunyavirales, and compares the morphological details of different viral structures imaged in the two types of samples. Advantages, disadvantages, and applications of conventional processing and HPF/FS are also presented and discussed.

ß-Cyclodextrins as affordable antivirals to treat coronavirus infection.

The SARS-CoV-2 pandemic made evident that there are only a few drugs against coronavirus. Here we aimed to identify a cost-effective antiviral with broad spectrum activity and high safety profile. Starting from a list of 116 drug candidates, we used molecular modelling tools to rank the 44 most promising inhibitors. Next, we tested their efficacy as antivirals against α and ß coronaviruses, such as the HCoV-229E and SARS-CoV-2 variants. Four drugs, OSW-1, U18666A, hydroxypropyl-ß-cyclodextrin (HßCD) and phytol, showed in vitro antiviral activity against HCoV-229E and SARS-CoV-2. The mechanism of action of these compounds was studied by transmission electron microscopy and by fusion assays measuring SARS-CoV-2 pseudoviral entry into target cells. Entry was inhibited by HßCD and U18666A, yet only HßCD inhibited SARS-CoV-2 replication in the pulmonary Calu-3 cells. Compared to the other cyclodextrins, ß-cyclodextrins were the most potent inhibitors, which interfered with viral fusion via cholesterol depletion. ß-cyclodextrins also prevented infection in a human nasal epithelium model ex vivo and had a prophylactic effect in the nasal epithelium of hamsters in vivo. All accumulated data point to ß-cyclodextrins as promising broad-spectrum antivirals against different SARS-CoV-2 variants and distant alphacoronaviruses. Given the wide use of ß-cyclodextrins for drug encapsulation and their high safety profile in humans, our results support their clinical testing as prophylactic antivirals.