Would It Be Possible to Stabilize Prefusion SARS-COV-2 Spikes with Ligands?

<p>Fusion to host cells and infection caused by Severe Acute Respiratory Syndrome coronavirus (SARS)-CoV2 was inhibited <i>in vitro</i> by PP mutations stabilizing prefusion states of their spike (S) protein native conformation, as reported by several authors. However, the possible stabilization of S by binding-ligands, rather than by mutations, have not been explored, nor it is yet known if it would be possible. In this work, the so called “spring-loaded switch-folding” (SLSF) expanding S amino acid residues 960-1010 was computationally targeted because SLSF surrounded the previously described PP mutations. The SLSF trimeric prefusion conformation consisted in 3x3 α-helices that require a transition to 3 longer α-helices before viral/host membrane fusion, similarly to what occurs in other enveloped viruses. Results of a double computational screening among hundred of thousands of natural compounds for binding to the wild-type isolated SLSF conformer predicted more leads for its trimers than for monomers. Further ranked by the number of SLSF-conformers bound, some of the predicted top-leads may deserve experimental validation. Additional screening among thousands of drugs identified Tinosorb, an star-shaped molecule, as the lowest binding-score lead to SLSF in the low nM range. However, despite its lower binding-score, 3-fold molecular symmetry and fitting the inner part of the SLSF α-helices, we were unable to experimentally show any specific inhibition of S-mediated membrane fusion using an VSV-pseudotyped infectivity assay, nor any virtual binding to S-SLSF using docking to whole native S trimers. Further exploring the star-shaped features may provide new molecular alternatives to cross-bind the α-helices of S-SLSF to hypothetically inhibit coronavirus fusion.<b></b></p>

Rapid inactivation of SARS-CoV-2 on copper touch surfaces determined using a cell culture infectivity assay

ABSTRACTCOVID-19, caused by SARS-CoV-2, was first reported in China in 2019 and has transmitted rapidly around the world, currently responsible for 83 million reported cases and over 1.8 million deaths. The mode of transmission is believed principally to be airborne exposure to respiratory droplets from symptomatic and asymptomatic patients but there is also a risk of the droplets contaminating fomites such as touch surfaces including door handles, stair rails etc, leading to hand pick up and transfer to eyes, nose and mouth. We have previously shown that human coronavirus 229E survives for more than 5 days on inanimate surfaces and another laboratory reproduced this for SARS-CoV-2 this year. However, we showed rapid inactivation of Hu-CoV-229E within 10 minutes on different copper surfaces while the other laboratory indicated this took 4 hours for SARS-CoV-2. So why the difference? We have repeated our work with SARS-CoV-2 and can confirm that this coronavirus can be inactivated on copper surfaces in as little as 1 minute. We discuss why the 4 hour result may be technically flawed.

A microfluidic impedance-based extended infectivity assay: combining retroviral amplification and cytopathic effect monitoring on a single lab-on-a-chip platform

We have established a lab-on-a-chip for detection, quantification and monitoring of virus – host cell interactions that are of great importance when evaluating the safety of pharmaceutical products.

Applicability of infectivity assay for the quantification of infectious human adenovirus genotype 5 in UV-irradiated wastewater

The use of infectivity assays in domestic wastewater samples are limited because of the concerns on cytotoxicity to host cells, thus, the UV inactivation efficiency of human adenoviruses (HAdVs) in wastewater remains unclear. In this study, a human adenovirus genotype five (HAdV-5) host cell line (A549 cells) was incubated with wastewater at varied dilutions from 1:1.5 to 1:4 (the ratio of wastewater to a mixture of wastewater and the cell culture medium) and the cytotoxicity was assessed by the cell morphology and viability. No change was observed in either cell viability or morphology in comparison with control samples, even at lowest dilution of 1:1.5, indicating the dilution allowed infectivity assays. The minimal degree of dilution to avoid cytotoxicity may differ with different water matrix. Consequently, the technique was applied to quantify spiked HAdV-5 after the UV disinfection of wastewater. A significant increase in UV disinfection efficiency was noted in wastewater and hydroxyl radicals (OH•) produced by the photosensitization of dissolved organic matter were suggested to be responsible for the enhancement. This study indicated that dilution can be a simple solution to avoid cytotoxicity, and UV inactivation may be enhanced in wastewater due to OH• radicals produced by UV radiation.

The Effect of GD1a Ganglioside-Expressing Bacterial Strains on Murine Norovirus Infectivity

In this study, we investigated the impact of GD1a-expressing bacterial strains on the infectivity of murine norovirus (MNV). Eligible bacterial strains were screened from a sewage sample using flow cytometry, and their genetic sequences of 16S rRNA were determined. The enzyme-linked immunosorbent assay (ELISA) was employed to analyze the binding between bacteria and MNV particles, and the plaque assay was used to assess the effects of GD1a-positive and negative strains on MNV infectivity. The result from ELISA shows that MNV particles are able to bind to both GD1a-positive and negative bacterial strains, but the binding to the GD1a-positive strain is more significant. The infectivity assay result further shows that the MNV infectious titer declined with an increasing concentration of GD1a-positive bacteria. The addition of anti-GD1a antibody in the infectivity assay led to the recovery of the MNV infectious titer, further confirming that the binding between MNV particles and bacterial GD1a ganglioside compromises MNV infectivity. Our findings highlight the role indigenous bacteria may play in the lifecycle of waterborne enteric viruses as well as the potential of exploiting them for virus transmission intervention and water safety improvement.

Biological Evaluation of Molecules of the azaBINOL Class as Antiviral Agents: Specific Inhibition of HIV-1 RNase H Activity by 7-Isopropoxy-8-(naphth-1-yl)quinoline

Inspired by bioactive biaryl-containing natural products found in plants and the marine environment, a series of synthetic compounds belonging to the azaBINOL chiral ligand family was evaluated for antiviral activity against HIV-1. Testing of 39 unique azaBINOLs in a singleround infectivity assay resulted in the identification of three promising antiviral compounds, including 7-isopropoxy-8-(naphth-1-yl)quinoline (azaBINOLB#24), which exhibited low-micromolar activity. The active compounds and several close structural analogues were further tested against three different HIV-1 envelope pseudotyped viruses as well as in a full-virus replication system (EASY-HIT). Mode-of-action studies using a time-of-addition assay indicated that azaBINOLB#24acts after viral entry but before viral assembly and budding. HIV-1 reverse transcriptase (RT) assays that individually test for polymerase and RNase H activity were used to demonstrate thatB#24inhibits RNase H activity, most likely allosterically. Further binding analysis using bio-layer interferometry (BLI) showed thatB#24interacts with HIV-1 RT in a highly specific manner. These results indicate that azaBINOLB#24is a potentially viable, novel lead for the development of new HIV-1 RNase H inhibitors. Furthermore, this study demonstrates that the survey of libraries of synthetic compounds, designed purely with the goal of facilitating chemical synthesis in mind, may yield unexpected and selective drug leads for the development of new antiviral agents.