IR spectroscopy is an excellent method for biological analyses. It enables the nonperturbative, label-free extraction of biochemical information and images toward diagnosis and the assessment of cell functionality. Although not strictly microscopy in the conventional sense, it allows the construction of images of tissue or cell architecture by the passing of spectral data through a variety of computational algorithms. Because such images are constructed from fingerprint spectra, the notion is that they can be an objective reflection of the underlying health status of the analyzed sample. One of the major difficulties in the field has been determining a consensus on spectral pre-processing and data analysis. This manuscript brings together as coauthors some of the leaders in this field to allow the standardization of methods and procedures for adapting a multistage approach to a methodology that can be applied to a variety of cell biological questions or used within a clinical setting for disease screening or diagnosis. We describe a protocol for collecting IR spectra and images from biological samples (e.g., fixed cytology and tissue sections, live cells or biofluids) that assesses the instrumental options available, appropriate sample preparation, different sampling modes as well as important advances in spectral data acquisition. After acquisition, data processing consists of a sequence of steps including quality control, spectral pre-processing, feature extraction and classification of the supervised or unsupervised type. A typical experiment can be completed and analyzed within hours. Example results are presented on the use of IR spectra combined with multivariate data processing.
Foot-and-mouth disease virus (FMDV) induces a very rapid inhibitionpro enabled mutant forms of the eIF4GII sequence to be generated that are differentially resistant to either one of these proteases. These results confirmed the specificity of each protease and showed that the mutant forms of the fusion protein substrate retained their correct sensitivity to other proteases.
The delivery of safe, visible wavelengths of light can be an effective, pathogen-agnostic, countermeasure that would expand the current portfolio of SARS-CoV-2 intervention strategies beyond the conventional approaches of vaccine, antibody, and antiviral therapeutics. Employing custom biological light units, that incorporate optically engineered light-emitting diode (LED) arrays, we harnessed monochromatic wavelengths of light for uniform delivery across biological surfaces. We demonstrated that primary 3D human tracheal/bronchial-derived epithelial tissues tolerated high doses of a narrow spectral band of visible light centered at a peak wavelength of 425 nm. We extended these studies to Vero E6 cells to understand how light may influence the viability of a mammalian cell line conventionally used for assaying SARS-CoV-2. The exposure of single-cell monolayers of Vero E6 cells to similar doses of 425 nm blue light resulted in viabilities that were dependent on dose and cell density. Doses of 425 nm blue light that are well-tolerated by Vero E6 cells also inhibited infection and replication of cell-associated SARS-CoV-2 by > 99% 24 h post-infection after a single five-minute light exposure. Moreover, the 425 nm blue light inactivated cell-free betacoronaviruses including SARS-CoV-1, MERS-CoV, and SARS-CoV-2 up to 99.99% in a dose-dependent manner. Importantly, clinically applicable doses of 425 nm blue light dramatically inhibited SARS-CoV-2 infection and replication in primary human 3D tracheal/bronchial tissue. Safe doses of visible light should be considered part of the strategic portfolio for the development of SARS-CoV-2 therapeutic countermeasures to mitigate coronavirus disease 2019 (COVID-19).
International audienceAvailable empirical data on the natural occurrence of ruminant pestiviruses has shown that bovine viral diarrhoea virus (BVDV) is nearly exclusively found in cattle, whereas both border disease virus (BDV) and BVDV can be isolated from sheep. During routine genetic typing of pestivirus RNA from UK cattle diagnosed as BVDV positive between 2006 and 2008, five samples that were classified as BDV positive yielded positive virus isolates in cell cultures. The samples originated from animals that had shown signs typical for BVD. Phylogenetic analysis of the bovine BDVs showed that two belonged to the BDV-1a group and three to the BDV-1b group, thereby matching the genetic diversity seen for previously described UK ovine BDVs. Antigenic typing with a set of monoclonal antibodies (MABs) showed that all bovine BDVs lacked one or more epitopes conserved among ovine BDV-1 isolates, and that they had gained reactivity with at least one BVDV-1 specific MAB. Serial passaging of two of the virus isolates in ovine cell cultures did not change the epitope expression pattern. These findings suggest that the presumed natural resistance of cattle against infection with BDV no longer holds. A consequence of this is that BVD diagnostic assays should be checked for their ability to also detect BDV, and also highlights the need for monitoring of the BDV status in sheep that may be in contact with cattle in areas with organised BVD control programmes
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