Search Results (4)

SARS-CoV-2 has been shown to lose the furin polybasic cleavage site (FCS) following adaptation on cell culture. Deletion occurring in this region, which may include also the FCS flanking regions, seem not to affect virus replication in vitro; however, a chimeric SARS-CoV-2 virus without the sole FCS motif has been associated with lower virulence in mice and lower neutralization values. Moreover, SARS-CoV-2 virus lacking the FCS was shed to lower titers from experimentally infected ferrets and was not transmitted to cohoused sentinel animals, unlike wild-type virus. In this study, we investigated the replication kinetics and cellular tropism of a SARS-CoV-2 isolate carrying a 10-amino acid deletion in the spike protein spanning the FCS in lung ex vivo organ cultures of mink. Furthermore, we tested the neutralization capabilities of human convalescent SARS-CoV-2 positive serum samples against this virus. We showed that this deletion did not significantly hamper neither ex vivo replication nor neutralization activity by convalescent serum samples. This study highlights the importance of the preliminary phenotypic characterization of emerging viruses in ex vivo models and demonstrates that mink lung tissues are permissive to the replication of a mutant form of SARS-CoV-2 showing a deletion spanning the FCS. Notably, we also highlight the need for sequencing viral stocks before any infection study as large deletions may occur leading to the misinterpretation of results. Full article

Biomedical research is multidisciplinary and often uses integrated approaches performing different experimental models with complementary functions. This approach is important to understand the pathogenetic mechanisms concerning the effects of environmental pollution on human health. The biological activity of the substances is investigated at least to three levels using molecular, cellular, and human tissue models. Each of these is able to give specific answers to experimental problems. A scientific approach, using biological methods (wet lab), cell cultures (cell lines or primary), isolated organs (three-dimensional cell cultures of primary epithelial cells), and animal organisms, including the human body, aimed to understand the effects of air pollution on the onset of diseases of the respiratory system. Biological methods are divided into three complementary models: in vitro, ex vivo, and in vivo. In vitro experiments do not require the use of whole organisms (in vivo study), while ex vivo experiments use isolated organs or parts of organs. The concept of complementarity and the informatic support are useful tools to organize, analyze, and interpret experimental data, with the aim of discussing scientific notions with objectivity and rationality in biology and medicine. In this scenario, the integrated and complementary use of different experimental models is important to obtain useful and global information that allows us to identify the effect of inhaled pollutants on the incidence of respiratory diseases in the exposed population. In this review, we focused our attention on the impact of air pollution in airway diseases with a rapid and descriptive analysis on the role of epithelium and on the experimental cell models useful to study the effect of toxicants on epithelial cells. Full article

Bovine herpesvirus-1 (BoHV-1) infection contributes to keratoconjunctivitis, respiratory disease, and reproductive losses in cattle. The objective of this study was to determine the most appropriate ophthalmic antiviral agent for BoHV-1 inhibition using in-vitro culture and novel ex-vivo bovine corneal modeling. Half-maximal inhibitory concentrations of BoHV-1 were determined for cidofovir, ganciclovir, idoxuridine, and trifluridine via in-vitro plaque reduction assays. In-vitro cytotoxicity was compared amongst these compounds via luciferase assays. Trifluridine and cidofovir were the most potent BoHV-1 inhibitors in vitro, while trifluridine and idoxuridine were the most cytotoxic agents. Therefore, cidofovir was the most potent non-cytotoxic agent and was employed in the ex-vivo corneal assay. Corneoscleral rings (n = 36) from fresh cadaver bovine globes were harvested and equally divided into an uninfected, untreated control group; a BoHV-1-infected, untreated group; and a BoHV-1-infected, cidofovir-treated group. Virus isolation for BoHV-1 titers was performed from corneal tissue and liquid media. Histologic measurements of corneal thickness, epithelial cell density, and tissue organization were compared between groups. Substantial BoHV-1 replication was observed in infected, untreated corneas, but BoHV-1 titer was significantly reduced in cidofovir-treated (1.69 ± 0.08 × 10³ PFU/mL) versus untreated (8.25 ± 0.25 × 10⁵ PFU/mL, p < 0.0001) tissues by day 2 of culture. No significant differences in histologic criteria were observed between groups. In conclusion, cidofovir warrants further investigation as treatment for BoHV-1 keratoconjunctivitis, with future studies needed to assess in-vivo tolerability and efficacy. Full article

The Gammacoronavirus infectious bronchitis virus (IBV) causes a highly contagious and economically important respiratory disease in poultry. In the laboratory, most IBV strains are restricted to replication in ex vivo organ cultures or in ovo and do not replicate in cell culture, making the study of their basic virology difficult. Entry of IBV into cells is facilitated by the large glycoprotein on the surface of the virion, the spike (S) protein, comprised of S1 and S2 subunits. Previous research showed that the S2′ cleavage site is responsible for the extended tropism of the IBV Beaudette strain. This study aims to investigate whether protease treatment can extend the tropism of other IBV strains. Here we demonstrate that the addition of exogenous trypsin during IBV propagation in cell culture results in significantly increased viral titres. Using a panel of IBV strains, exhibiting varied tropisms, the effects of spike cleavage on entry and replication were assessed by serial passage cell culture in the presence of trypsin. Replication could be maintained over serial passages, indicating that the addition of exogenous protease is sufficient to overcome the barrier to infection. Mutations were identified in both S1 and S2 subunits following serial passage in cell culture. This work provides a proof of concept that exogenous proteases can remove the barrier to IBV replication in otherwise non-permissive cells, providing a platform for further study of elusive field strains and enabling sustainable vaccine production in vitro. Full article