Inhibition of SARS-CoV-2 Infection in Vero Cells by Bovine Lactoferrin under Different Iron-Saturation States.

Despite the rapid mass vaccination against COVID-19, the emergence of new SARS-CoV-2 variants of concern, such as omicron, is still a great distress, and new therapeutic options are needed. Bovine lactoferrin (bLf), a multifunctional iron-binding glycoprotein available in unsaturated (apo-bLf) and saturated (holo-bLf) forms, has been shown to exert broad-spectrum antiviral activity against many viruses. In this study, we evaluated the efficacy of both forms of bLf at 1 mg/mL against infection of Vero cells by SARS-CoV-2. As assessed with antiviral assays, an equivalent significant reduction in virus infection by about 70% was observed when either form of bLf was present throughout the infection procedure with the SARS-CoV-2 ancestral or omicron strain. This inhibitory effect seemed to be concentrated during the early steps of virus infection, since a significant reduction in its efficiency by about 60% was observed when apo- or holo-bLf were incubated with the cells before or during virus addition, with no significant difference between the antiviral effects of the distinct iron-saturation states of the protein. However, an ultrastructural analysis of bLf treatment during the early steps of virus infection revealed that holo-bLf was somewhat more effective than apo-bLf in inhibiting virus entry. Together, these data suggest that bLf mainly acts in the early events of SARS-CoV-2 infection and is effective against the ancestral virus as well as its omicron variant. Considering that there are no effective treatments to COVID-19 with tolerable toxicity yet, bLf shows up as a promising candidate.

Stability of different influenza subtypes: How can high hydrostatic pressure be a useful tool for vaccine development?

BACKGROUND: Avian influenza A viruses can cross naturally into mammals and cause severe diseases, as observed for H5N1. The high lethality of human infections causes major concerns about the real risk of a possible pandemic of severe diseases to which human susceptibility may be high and universal. High hydrostatic pressure (HHP) is a valuable tool for studies regarding the folding of proteins and the assembly of macromolecular structures such as viruses; furthermore, HHP has already been demonstrated to promote viral inactivation. METHODS: Here, we investigated the structural stability of avian and human influenza viruses using spectroscopic and light-scattering techniques. We found that both particles have similar structural stabilities and that HHP promotes structural changes. RESULTS: HHP induced slight structural changes to both human and avian influenza viruses, and these changes were largely reversible when the pressure returned to its initial level. The spectroscopic data showed that H3N2 was more pressure-sensitive than H3N8. Structural changes did not predict changes in protein function, as H3N2 fusion activity was not affected, while H3N8 fusion activity drastically decreased. The fusion activity of H1N1 was also strongly affected by HHP. In all cases, HHP caused inactivation of the different influenza viruses. CONCLUSIONS: HHP may be a useful tool for vaccine development, as it induces minor and reversible structural changes that may be associated with partial preservation of viral biological activities and may potentiate their immunogenic response while abolishing their infectivity. We also confirmed that, although pressure does not promote drastic changes in viral particle structure, it can distinctly affect viral fusion activity.

The structural dynamics of the flavivirus fusion peptide-membrane interaction.

Membrane fusion is a crucial step in flavivirus infections and a potential target for antiviral strategies. Lipids and proteins play cooperative roles in the fusion process, which is triggered by the acidic pH inside the endosome. This acidic environment induces many changes in glycoprotein conformation and allows the action of a highly conserved hydrophobic sequence, the fusion peptide (FP). Despite the large volume of information available on the virus-triggered fusion process, little is known regarding the mechanisms behind flavivirus-cell membrane fusion. Here, we evaluated the contribution of a natural single amino acid difference on two flavivirus FPs, FLA(G) ((98)DRGWGNGCGLFGK(110)) and FLA(H) ((98)DRGWGNHCGLFGK(110)), and investigated the role of the charge of the target membrane on the fusion process. We used an in silico approach to simulate the interaction of the FPs with a lipid bilayer in a complementary way and used spectroscopic approaches to collect conformation information. We found that both peptides interact with neutral and anionic micelles, and molecular dynamics (MD) simulations showed the interaction of the FPs with the lipid bilayer. The participation of the indole ring of Trp appeared to be important for the anchoring of both peptides in the membrane model, as indicated by MD simulations and spectroscopic analyses. Mild differences between FLA(G) and FLA(H) were observed according to the pH and the charge of the target membrane model. The MD simulations of the membrane showed that both peptides adopted a bend structure, and an interaction between the aromatic residues was strongly suggested, which was also observed by circular dichroism in the presence of micelles. As the FPs of viral fusion proteins play a key role in the mechanism of viral fusion, understanding the interactions between peptides and membranes is crucial for medical science and biology and may contribute to the design of new antiviral drugs.