Traceability of SARS-CoV-2 transmission through quasispecies analysis.

During COVID-19 pandemic, consensus genomic sequences were used for rapidly monitor the spread of the virus worldwide. However, less attention was paid to intrahost genetic diversity. In fact, in the infected host, SARS-CoV-2 consists in an ensemble of replicating and closely related viral variants so-called quasispecies. Here we show that intrahost single nucleotide variants (iSNVs) represent a target for contact tracing analysis. Our data indicate that in the acute phase of infection, in highly likely transmission links, the number of viral particles transmitted from one host to another (bottleneck size) is large enough to propagate iSNVs among individuals. Furthermore, we demonstrate that, during SARS-CoV-2 outbreaks when the consensus sequences are identical, it is possible to reconstruct the transmission chains by genomic investigations of iSNVs. Specifically, we found that it is possible to identify transmission chains by limiting the analysis of iSNVs to only three well-conserved genes, namely nsp2, ORF3, and ORF7.

DHFR Inhibitors Display a Pleiotropic Anti-Viral Activity against SARS-CoV-2: Insights into the Mechanisms of Action.

During the COVID-19 pandemic, drug repurposing represented an effective strategy to obtain quick answers to medical emergencies. Based on previous data on methotrexate (MTX), we evaluated the anti-viral activity of several DHFR inhibitors in two cell lines. We observed that this class of compounds showed a significant influence on the virus-induced cytopathic effect (CPE) partly attributed to the intrinsic anti-metabolic activity of these drugs, but also to a specific anti-viral function. To elucidate the molecular mechanisms, we took advantage of our EXSCALATE platform for in-silico molecular modelling and further validated the influence of these inhibitors on nsp13 and viral entry. Interestingly, pralatrexate and trimetrexate showed superior effects in counteracting the viral infection compared to other DHFR inhibitors. Our results indicate that their higher activity is due to their polypharmacological and pleiotropic profile. These compounds can thus potentially give a clinical advantage in the management of SARS-CoV-2 infection in patients already treated with this class of drugs.

SARS-CoV-2 Systemic Effects: New Clues.

To date, much discussion has been had on severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) lung infection associated with COVID-19 onset, of which the major manifestation is characterized by a "cytokine storm" [...].

Virucidal efficacy of a novel silver-based disinfectant against SARS-CoV-2 Omicron BA.5.

In this study we evaluated the antiviral activity of the Silver Barrier® disinfectant against SARSCoV-2. Silver Barrier® showed time- and concentration-dependent antiviral activity against SARSCoV-2. After 5 min contact time, Silver Barrier® at 0.002% showed a strong inhibitory effect (p<0.001), with a 2-fold reduction of viral genome copy numbers, and a robust suppression (94%) of SARS-CoV-2 infectivity. Considering the effects obtained in solution and within a very short time, Silver Barrier® stands as an excellent new candidate for the disinfection of work environments, especially at the healthcare level, where there are people at high risk of serious illnesses.

The D405N Mutation in the Spike Protein of SARS-CoV-2 Omicron BA.5 Inhibits Spike/Integrins Interaction and Viral Infection of Human Lung Microvascular Endothelial Cells.

Severe COVID-19 is characterized by angiogenic features, such as intussusceptive angiogenesis, endothelialitis, and activation of procoagulant pathways. This pathological state can be ascribed to a direct SARS-CoV-2 infection of human lung ECs. Recently, we showed the capability of SARS-CoV-2 to infect ACE2-negative primary human lung microvascular endothelial cells (HL-mECs). This occurred through the interaction of an Arg-Gly-Asp (RGD) motif, endowed on the Spike protein at position 403-405, with αvß3 integrin expressed on HL-mECs. HL-mEC infection promoted the remodeling of cells toward a pro-inflammatory and pro-angiogenic phenotype. The RGD motif is distinctive of SARS-CoV-2 Spike proteins up to the Omicron BA.1 subvariant. Suddenly, a dominant D405N mutation was expressed on the Spike of the most recently emerged Omicron BA.2, BA.4, and BA.5 subvariants. Here we demonstrate that the D405N mutation inhibits Omicron BA.5 infection of HL-mECs and their dysfunction because of the lack of Spike/integrins interaction. The key role of ECs in SARS-CoV-2 pathogenesis has been definitively proven. Evidence of mutations retrieving the capability of SARS-CoV-2 to infect HL-mECs highlights a new scenario for patients infected with the newly emerged SARS-CoV-2 Omicron subvariants, suggesting that they may display less severe disease manifestations than those observed with previous variants.

Detection of SARS-CoV-2 Alpha variant in a severely immunocompromised HIV-1-infected patient in the omicron era.

Persistence of detectable viral RNA does not depend on the symptomatic status of the patients. Here we describe the case of a strongly immunocompromised patient living with a prolonged SARS-CoV-2 Alpha variant infection without showing any symptoms. The importance of our findings is that the persistence of infection with an old SARS-CoV-2 strain, in an immunocompromised host, may allow recombination events generating new viral variants whose pathogenicity cannot be predicted. Our observation calls for the urgent need for continuous monitoring of SARS-CoV-2 genomic evolution in immunocompromised patients.

Genetic and Structural Data on the SARS-CoV-2 Omicron BQ.1 Variant Reveal Its Low Potential for Epidemiological Expansion.

The BQ.1 SARS-CoV-2 variant, also known as Cerberus, is one of the most recent Omicron descendant lineages. Compared to its direct progenitor BA.5, BQ.1 has some additional spike mutations in some key antigenic sites, which confer further immune escape ability over other circulating lineages. In such a context, here, we perform a genome-based survey aimed at obtaining a complete-as-possible nuance of this rapidly evolving Omicron subvariant. Genetic data suggest that BQ.1 represents an evolutionary blind background, lacking the rapid diversification that is typical of a dangerous lineage. Indeed, the evolutionary rate of BQ.1 is very similar to that of BA.5 (7.6 × 10-4 and 7 × 10-4 subs/site/year, respectively), which has been circulating for several months. The Bayesian Skyline Plot reconstruction indicates a low level of genetic variability, suggesting that the peak was reached around 3 September 2022. Concerning the affinity for ACE2, structure analyses (also performed by comparing the properties of BQ.1 and BA.5 RBD) indicate that the impact of the BQ.1 mutations may be modest. Likewise, immunoinformatic analyses showed moderate differences between the BQ.1 and BA5 potential B-cell epitopes. In conclusion, genetic and structural analyses on SARS-CoV-2 BQ.1 suggest no evidence of a particularly dangerous or high expansion capability. Genome-based monitoring must continue uninterrupted for a better understanding of its descendants and all other lineages.

Competition for dominance within replicating quasispecies during prolonged SARS-CoV-2 infection in an immunocompromised host.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants of concern (VOCs) emerge for their capability to better adapt to the human host aimed and enhance human-to-human transmission. Mutations in spike largely contributed to adaptation. Viral persistence is a prerequisite for intra-host virus evolution, and this likely occurred in immunocompromised patients who allow intra-host long-term viral replication. The underlying mechanism leading to the emergence of variants during viral persistence in the immunocompromised host is still unknown. Here, we show the existence of an ensemble of minor mutants in the early biological samples obtained from an immunocompromised patient and their dynamic interplay with the master mutant during a persistent and productive long-term infection. In particular, after 222 days of active viral replication, the original master mutant, named MB610, was replaced by a minor quasispecies (MB61222) expressing two critical mutations in spike, namely Q493K and N501T. Isolation of the two viruses allowed us to show that MB61222 entry into target cells occurred mainly by the fusion at the plasma membrane (PM), whereas endocytosis characterized the entry mechanism used by MB610. Interestingly, coinfection of two human cell lines of different origin with the SARS-CoV-2 isolates highlighted the early and dramatic predominance of MB61222 over MB610 replication. This finding may be explained by a faster replicative activity of MB61222 as compared to MB610 as well as by the capability of MB61222 to induce peculiar viral RNA-sensing mechanisms leading to an increased production of interferons (IFNs) and, in particular, of IFN-induced transmembrane protein 1 (IFITM1) and IFITM2. Indeed, it has been recently shown that IFITM2 is able to restrict SARS-CoV-2 entry occurring by endocytosis. In this regard, MB61222 may escape the antiviral activity of IFITMs by using the PM fusion pathway for entry into the target cell, whereas MB610 cannot escape this host antiviral response during MB61222 coinfection, since it has endocytosis as the main pathway of entry. Altogether, our data support the evidence of quasispecies fighting for host dominance by taking benefit from the cell machinery to restrict the productive infection of competitors in the viral ensemble. This finding may explain, at least in part, the extraordinary rapid worldwide turnover of VOCs that use the PM fusion pathway to enter into target cells over the original pandemic strain.

Genomic surveillance of SARS-CoV-2 in patients presenting neurological manifestations.

During the first wave of infections, neurological symptoms in Coronavirus Disease 2019 (COVID-19) patients raised particular concern, suggesting that, in a subset of patients, the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) could invade and damage cells of the central nervous system (CNS). Indeed, up to date several in vitro and in vivo studies have shown the ability of SARS-CoV-2 to reach the CNS. Both viral and/or host related features could explain why this occurs only in certain individuals and not in all the infected population. The aim of the present study was to evaluate if onset of neurological manifestations in COVID-19 patients was related to specific viral genomic signatures. To this end, viral genome was extracted directly from nasopharyngeal swabs of selected SARS-CoV-2 positive patients presenting a spectrum of neurological symptoms related to COVID-19, ranging from anosmia/ageusia to more severe symptoms. By adopting a whole genome sequences approach, here we describe a panel of known as well as unknown mutations detected in the analyzed SARS-CoV-2 genomes. While some of the found mutations were already associated with an improved viral fitness, no common signatures were detected when comparing viral sequences belonging to specific groups of patients. In conclusion, our data support the notion that COVID-19 neurological manifestations are mainly linked to patient-specific features more than to virus genomic peculiarities.

Peptide-Antibody Fusions Engineered by Phage Display Exhibit an Ultrapotent and Broad Neutralization of SARS-CoV-2 Variants.

The spread of COVID-19 has been exacerbated by the emergence of variants of concern (VoC). Many VoC contain mutations in the spike protein (S-protein) and are implicated in infection and response to therapeutics. Bivalent neutralizing antibodies (nAbs) targeting the S-protein receptor-binding domain (RBD) are promising therapeutics for COVID-19, but they are limited by low potency and vulnerability to RBD mutations in VoC. To address these issues, we used naïve phage-displayed peptide libraries to isolate and optimize 16-residue peptides that bind to the RBD or the N-terminal domain (NTD) of the S-protein. We fused these peptides to the N-terminus of a moderate-affinity nAb to generate tetravalent peptide-IgG fusions, and we showed that both classes of peptides were able to improve affinities for the S-protein trimer by >100-fold (apparent KD < 1 pM). Critically, cell-based infection assays with a panel of six SARS-CoV-2 variants demonstrated that an RBD-binding peptide was able to enhance the neutralization potency of a high-affinity nAb >100-fold. Moreover, this peptide-IgG was able to neutralize variants that were resistant to the same nAb in the bivalent IgG format, including the dominant B.1.1.529 (Omicron) variant that is resistant to most clinically approved therapeutic nAbs. To show that this approach is general, we fused the same peptide to a clinically approved nAb drug and showed that it enabled the neutralization of a resistant variant. Taken together, these results establish minimal peptide fusions as a modular means to greatly enhance affinities, potencies, and breadth of coverage of nAbs as therapeutics for SARS-CoV-2.

Characterization of raloxifene as a potential pharmacological agent against SARS-CoV-2 and its variants.

The new coronavirus SARS-CoV-2 is the causative agent of the COVID-19 pandemic, which so far has caused over 6 million deaths in 2 years, despite new vaccines and antiviral medications. Drug repurposing, an approach for the potential application of existing pharmaceutical products to new therapeutic indications, could be an effective strategy to obtain quick answers to medical emergencies. Following a virtual screening campaign on the most relevant viral proteins, we identified the drug raloxifene, a known Selective Estrogen Receptor Modulator (SERM), as a new potential agent to treat mild-to-moderate COVID-19 patients. In this paper we report a comprehensive pharmacological characterization of raloxifene in relevant in vitro models of COVID-19, specifically in Vero E6 and Calu-3 cell lines infected with SARS-CoV-2. A large panel of the most common SARS-CoV-2 variants isolated in Europe, United Kingdom, Brazil, South Africa and India was tested to demonstrate the drug's ability in contrasting the viral cytopathic effect (CPE). Literature data support a beneficial effect by raloxifene against the viral infection due to its ability to interact with viral proteins and activate protective estrogen receptor-mediated mechanisms in the host cells. Mechanistic studies here reported confirm the significant affinity of raloxifene for the Spike protein, as predicted by in silico studies, and show that the drug treatment does not directly affect Spike/ACE2 interaction or viral internalization in infected cell lines. Interestingly, raloxifene can counteract Spike-mediated ADAM17 activation in human pulmonary cells, thus providing new insights on its mechanism of action. A clinical study in mild to moderate COVID-19 patients (NCT05172050) has been recently completed. Our contribution to evaluate raloxifene results on SARS-CoV-2 variants, and the interpretation of the mechanisms of action will be key elements to better understand the trial results, and to design new clinical studies aiming to evaluate the potential development of raloxifene in this indication.

Ultrapotent and broad neutralization of SARS-CoV-2 variants by modular, tetravalent, bi-paratopic antibodies.

Neutralizing antibodies (nAbs) that target the SARS-CoV-2 spike protein have received emergency use approval for treatment of COVID-19. However, with the emergence of variants of concern, there is a need for new treatment options. We report a format that enables modular assembly of bi-paratopic tetravalent nAbs with antigen-binding sites from two distinct nAbs. The tetravalent nAb purifies in high yield and exhibits biophysical characteristics that are comparable to those of clinically used therapeutic antibodies. The tetravalent nAb binds to the spike protein trimer at least 100-fold more tightly than bivalent IgGs (apparent KD < 1 pM) and neutralizes a broad array of SARS-CoV-2 pseudoviruses, chimeric viruses, and authentic viral variants with high potency. Together, these results establish the tetravalent diabody-Fc-Fab as a robust, modular platform for rapid production of drug-grade nAbs with potencies and breadth of coverage that greatly exceed those of conventional bivalent IgGs.

SARS-CoV-2 Infects Human ACE2-Negative Endothelial Cells through an αvß3 Integrin-Mediated Endocytosis Even in the Presence of Vaccine-Elicited Neutralizing Antibodies.

Integrins represent a gateway of entry for many viruses and the Arg-Gly-Asp (RGD) motif is the smallest sequence necessary for proteins to bind integrins. All Severe Acute Respiratory Syndrome Virus type 2 (SARS-CoV-2) lineages own an RGD motif (aa 403-405) in their receptor binding domain (RBD). We recently showed that SARS-CoV-2 gains access into primary human lung microvascular endothelial cells (HL-mECs) lacking Angiotensin-converting enzyme 2 (ACE2) expression through this conserved RGD motif. Following its entry, SARS-CoV-2 remodels cell phenotype and promotes angiogenesis in the absence of productive viral replication. Here, we highlight the αvß3 integrin as the main molecule responsible for SARS-CoV-2 infection of HL-mECs via a clathrin-dependent endocytosis. Indeed, pretreatment of virus with αvß3 integrin or pretreatment of cells with a monoclonal antibody against αvß3 integrin was found to inhibit SARS-CoV-2 entry into HL-mECs. Surprisingly, the anti-Spike antibodies evoked by vaccination were neither able to impair Spike/integrin interaction nor to prevent SARS-CoV-2 entry into HL-mECs. Our data highlight the RGD motif in the Spike protein as a functional constraint aimed to maintain the interaction of the viral envelope with integrins. At the same time, our evidences call for the need of intervention strategies aimed to neutralize the SARS-CoV-2 integrin-mediated infection of ACE2-negative cells in the vaccine era.

SARS-CoV-2 Infects Human ACE2-Negative Endothelial Cells through an αvβ3 Integrin-Mediated Endocytosis Even in the Presence of Vaccine-Elicited Neutralizing Antibodies

Integrins represent a gateway of entry for many viruses and the Arg-Gly-Asp (RGD) motif is the smallest sequence necessary for proteins to bind integrins. All Severe Acute Respiratory Syndrome Virus type 2 (SARS-CoV-2) lineages own an RGD motif (aa 403–405) in their receptor binding domain (RBD). We recently showed that SARS-CoV-2 gains access into primary human lung microvascular endothelial cells (HL-mECs) lacking Angiotensin-converting enzyme 2 (ACE2) expression through this conserved RGD motif. Following its entry, SARS-CoV-2 remodels cell phenotype and promotes angiogenesis in the absence of productive viral replication. Here, we highlight the αvβ3 integrin as the main molecule responsible for SARS-CoV-2 infection of HL-mECs via a clathrin-dependent endocytosis. Indeed, pretreatment of virus with αvβ3 integrin or pretreatment of cells with a monoclonal antibody against αvβ3 integrin was found to inhibit SARS-CoV-2 entry into HL-mECs. Surprisingly, the anti-Spike antibodies evoked by vaccination were neither able to impair Spike/integrin interaction nor to prevent SARS-CoV-2 entry into HL-mECs. Our data highlight the RGD motif in the Spike protein as a functional constraint aimed to maintain the interaction of the viral envelope with integrins. At the same time, our evidences call for the need of intervention strategies aimed to neutralize the SARS-CoV-2 integrin-mediated infection of ACE2-negative cells in the vaccine era.

SARS-CoV-2 AY.4.2 variant circulating in Italy: Genomic preliminary insight.

The appearance of emerging variants of SARS-CoV-2 carrying mutations into the spike protein has recently raised concern with respect to tracking their transmission and mitigating the impact in the evolving pandemic across countries. AY.4.2, a recently detected Delta variant sublineage, is considered a new variant under investigation (VUI) as it carries specific genetic signatures present in the spike protein, called Y145H and A222V. Here, using genomic epidemiology, we provide the first preliminary insight regarding the circulation of this emerging VUI in Italy.

Role of Q675H Mutation in Improving SARS-CoV-2 Spike Interaction with the Furin Binding Pocket.

Genotype screening was implemented in Italy and showed a significant prevalence of new SARS-CoV-2 mutants carrying Q675H mutation, near the furin cleavage site of spike protein. Currently, this mutation, which is expressed on different SARS-CoV-2 lineages circulating worldwide, has not been thoughtfully investigated. Therefore, we performed phylogenetic and biocomputational analysis to better understand SARS-CoV-2 Q675H mutants' evolutionary relationships with other circulating lineages and Q675H function in its molecular context. Our studies reveal that Q675H spike mutation is the result of parallel evolution because it arose independently in separate evolutionary clades. In silico data show that the Q675H mutation gives rise to a hydrogen-bonds network in the spike polar region. This results in an optimized directionality of arginine residues involved in interaction of spike with the furin binding pocket, thus improving proteolytic exposure of the viral protein. Furin was predicted to have a greater affinity for Q675H than Q675 substrate conformations. As a consequence, Q675H mutation could confer a fitness advantage to SARS-CoV-2 by promoting a more efficient viral entry. Interestingly, here we have shown that Q675H spike mutation is documented in all the VOCs. This finding highlights that VOCs are still evolving to enhance viral fitness and to adapt to the human host. At the same time, it may suggest Q675H spike mutation involvement in SARS-CoV-2 evolution.

Transparent Polymeric Formulations Effective against SARS-CoV-2 Infection.

The main route of the transmission of the SARS-CoV-2 virus is through airborne small aerosol particles containing viable virus as well as through droplets transmitted between people within close proximity. Transmission via contaminated surfaces has also been recognized as an important route for the spread of SARS-CoV-2 coronavirus. Among a variety of antimicrobial agents currently in use, polymers represent a class of biocides that have become increasingly important as an alternative to existing biocidal approaches. Two transparent polymeric compounds, containing silver and benzalkonium ions electrostatically bound to a polystyrene sulfonate backbone, were synthesized, through simple procedures, and evaluated for their antimicrobial properties against Gram-positive and Gram-negative bacteria and Candida albicans (ISO EN 1276) and for their antiviral activity toward 229E and SARS-CoV-2 coronaviruses (ISO UNI EN 14476:2019). The results showed that the two tested formulations are able to inhibit the growth of (1.5-5.5) × 1011 CFU of Gram-positive bacteria, Gram-negative bacteria, and of the fungal species Candida albicans. Both compounds were able to control the 229E and SARS-CoV-2 infection of a target cell in a time contact of 5 min, with a virucidal effect from 24 to 72 h postinfection, according to the European Medicines Agency (EMA) guidelines, where a product is considered virucidal upon achieving a reduction of 4 logarithms. This study observed a decrease of more than 5 logarithms, which implies that these formulations are likely ideal candidates for the realization of transparent surface coatings that are capable of maintaining remarkable antibacterial activity and SARS-CoV-2 antiviral properties over time.