DYRK1A promotes viral entry of highly pathogenic human coronaviruses in a kinase-independent manner.

Identifying host genes essential for Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) has the potential to reveal novel drug targets and further our understanding of Coronavirus Disease 2019 (COVID-19). We previously performed a genome-wide CRISPR/Cas9 screen to identify proviral host factors for highly pathogenic human coronaviruses. Few host factors were required by diverse coronaviruses across multiple cell types, but DYRK1A was one such exception. Although its role in coronavirus infection was previously undescribed, DYRK1A encodes Dual Specificity Tyrosine Phosphorylation Regulated Kinase 1A and is known to regulate cell proliferation and neuronal development. Here, we demonstrate that DYRK1A regulates ACE2 and DPP4 transcription independent of its catalytic kinase function to support SARS-CoV, SARS-CoV-2, and Middle East Respiratory Syndrome Coronavirus (MERS-CoV) entry. We show that DYRK1A promotes DNA accessibility at the ACE2 promoter and a putative distal enhancer, facilitating transcription and gene expression. Finally, we validate that the proviral activity of DYRK1A is conserved across species using cells of nonhuman primate and human origin. In summary, we report that DYRK1A is a novel regulator of ACE2 and DPP4 expression that may dictate susceptibility to multiple highly pathogenic human coronaviruses.

[Antiviral activity of basidial fungus Inonotus obliquus aqueous extract against SARS-CоV-2 virus (Coronaviridae: Betacoronavirus: Sarbecovirus) in vivo in BALB/c mice model].

INTRODUCTION: The COVID-19 pandemic combined with seasonal epidemics of respiratory viral diseases requires targeted antiviral prophylaxis with restorative and immunostimulant drugs. The compounds of natural origin are low-toxic, but active against several viruses at the same time. One of the most famous compounds is Inonotus obliquus aqueous extract. The fruit body of basidial fungus I. obliquus is called Chaga mushroom. The aim of the work ‒ was to study the antiviral activity of I. obliquus aqueous extract against the SARS-CoV-2 virus in vivo. MATERIALS AND METHODS: Antiviral activity of I. obliquus aqueous extract sample (#20-17) was analyzed against strain of SARS-CoV-2 Omicron ВА.5.2 virus. The experiments were carried out in BALB/c inbred mice. The SARS-CoV-2 viral load was measured using quantitative real-time PCR combined with reverse transcription. The severity of lung tissue damage was assessed by histological methods. RESULTS: The peak values of the viral load in murine lung tissues were determined 72 hours after intranasal inoculation at dose of 2,85 lg TCID50. The quantitative real-time PCR testing has shown a significant decrease in the viral load compared to the control group by 4,65 lg copies/ml and 5,72 lg copies/ml in the lung tissue and nasal cavity samples, respectively. Histological methods revealed that the decrease in the number and frequency of observed pathomorphological changes in murine lung tissues depended on the introduction of the compound under study. CONCLUSION: The results obtained indicate the possibility of using basidial fungus Inonotus obliquus aqueous extract as a preventive agent against circulating variants of SARS-CoV-2 virus.

[Evaluation of the dynamics of detection of viable SARS-CoV-2 (Coronaviridae: Betacoronavirus: Sarbecovirus) in biological samples obtained from patients with COVID-19 in a health care setting, as one of the indicators of the infectivity of the virus].

INTRODUCTION: The study of the mechanisms of transmission of the SARS-CoV-2 virus is the basis for building a strategy for anti-epidemic measures in the context of the COVID-19 pandemic. Understanding in what time frame a patient can spread SARS-CoV-2 is just as important as knowing the transmission mechanisms themselves. This information is necessary to develop effective measures to prevent infection by breaking the chains of transmission of the virus. The aim of the work is to identify the infectious SARS-CoV-2 virus in patient samples in the course of the disease and to determine the duration of virus shedding in patients with varying severity of COVID-19. MATERIALS AND METHODS: In patients included in the study, biomaterial (nasopharyngeal swabs) was subjected to analysis by quantitative RT-PCR and virological determination of infectivity of the virus. RESULTS: We have determined the timeframe of maintaining the infectivity of the virus in patients hospitalized with severe and moderate COVID-19. Based on the results of the study, we made an analysis of the relationship between the amount of detected SARS-CoV-2 RNA and the infectivity of the virus in vitro in patients with COVID-19. The median time of the infectious virus shedding was 8 days. In addition, a comparative analysis of different protocols for the detection of the viral RNA in relation to the identification of the infectious virus was carried out. CONCLUSION: The obtained data make it possible to assess the dynamics of SARS-CoV-2 detection and viral load in patients with COVID-19 and indicate the significance of these parameters for the subsequent spread of the virus and the organization of preventive measures.

A multi-specific, multi-affinity antibody platform neutralizes sarbecoviruses and confers protection against SARS-CoV-2 in vivo.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative agent of coronavirus disease 2019 (COVID-19), has been responsible for a global pandemic. Monoclonal antibodies (mAbs) have been used as antiviral therapeutics; however, these therapeutics have been limited in efficacy by viral sequence variability in emerging variants of concern (VOCs) and in deployment by the need for high doses. In this study, we leveraged the multi-specific, multi-affinity antibody (Multabody, MB) platform, derived from the human apoferritin protomer, to enable the multimerization of antibody fragments. MBs were shown to be highly potent, neutralizing SARS-CoV-2 at lower concentrations than their corresponding mAb counterparts. In mice infected with SARS-CoV-2, a tri-specific MB targeting three regions within the SARS-CoV-2 receptor binding domain was protective at a 30-fold lower dose than a cocktail of the corresponding mAbs. Furthermore, we showed in vitro that mono-specific MBs potently neutralize SARS-CoV-2 VOCs by leveraging augmented avidity, even when corresponding mAbs lose their ability to neutralize potently, and that tri-specific MBs expanded the neutralization breadth beyond SARS-CoV-2 to other sarbecoviruses. Our work demonstrates how avidity and multi-specificity combined can be leveraged to confer protection and resilience against viral diversity that exceeds that of traditional monoclonal antibody therapies.

An immediate way to lower pandemic risk: (not) seizing the low-hanging fruit (bat).

What is the least that humanity can do to mitigate the risks of future pandemics, to prevent worldwide surges in human deaths, illness, and suffering-and more waves of multitrillion US dollar impacts on the global economy? The issues around our consumption and trading of wildlife are diverse and complex, with many rural communities being dependent on wild meat for their nutritional needs. But bats might be one taxonomic group that can be successfully eliminated from the human diet and other uses, with minimal costs or inconvenience to the vast majority of the 8 billion people on Earth. The order Chiroptera merits genuine respect given all that these species contribute to human food supplies through pollination services provided by the frugivores and to disease risk mitigation delivered by insectivorous species. The global community missed its chance to stop SARS-CoV and SARS-CoV-2 from emerging-how many more times will humanity allow this cycle to repeat? How long will governments ignore the science that is in front of them? It's past time for humans to do the least that can be done. A global taboo is needed whereby humanity agrees to leave bats alone, not fear them or try to chase them away or cull them, but to let them have the habitats they need and live undisturbed by humans.

Different electrostatic forces drive the binding kinetics of SARS-CoV, SARS-CoV-2 and MERS-CoV Envelope proteins with the PDZ2 domain of ZO1.

The Envelope protein (E) is a structural protein encoded by the genome of SARS-CoV, SARS-CoV-2 and MERS-CoV Coronaviruses. It is poorly present in the virus but highly expressed in the host cell, with prominent role in virus assembly and virulence. The E protein possesses a PDZ-binding motif (PBM) at its C terminus that allows it to interact with host PDZ domain containing proteins. ZO1 is a key protein in assembling the cytoplasmic plaque of epithelial and endothelial Tight Junctions (TJs) as well as in determining cell differentiation, proliferation and polarity. The PDZ2 domain of ZO1 is known to interact with the Coronaviruses Envelope proteins, however the molecular details of such interaction have not been established. In this paper we directly measured, through Fluorescence Resonance Energy Transfer and Stopped-Flow methodology, the binding kinetics of the PDZ2 domain of ZO1 with peptides mimicking the C-terminal portion of the Envelope protein from SARS-CoV, SARS-CoV-2 and MERS-CoV in different ionic strength conditions. Interestingly, the peptide mimicking the E protein from MERS-CoV display much higher microscopic association rate constant with PDZ2 compared to SARS-CoV and SARS-CoV-2 suggesting a stronger contribution of electrostatic forces in the early events of binding. A comparison of thermodynamic and kinetic data obtained at increasing ionic strengths put in evidence different contribution of electrostatics in the recognition and complex formation events for the three peptides. Our data are discussed under the light of available structural data of PDZ2 domain of ZO1 and of previous works about these protein systems.

Spike substitution T813S increases Sarbecovirus fusogenicity by enhancing the usage of TMPRSS2.

SARS-CoV Spike (S) protein shares considerable homology with SARS-CoV-2 S, especially in the conserved S2 subunit (S2). S protein mediates coronavirus receptor binding and membrane fusion, and the latter activity can greatly influence coronavirus infection. We observed that SARS-CoV S is less effective in inducing membrane fusion compared with SARS-CoV-2 S. We identify that S813T mutation is sufficient in S2 interfering with the cleavage of SARS-CoV-2 S by TMPRSS2, reducing spike fusogenicity and pseudoparticle entry. Conversely, the mutation of T813S in SARS-CoV S increased fusion ability and viral replication. Our data suggested that residue 813 in the S was critical for the proteolytic activation, and the change from threonine to serine at 813 position might be an evolutionary feature adopted by SARS-2-related viruses. This finding deepened the understanding of Spike fusogenicity and could provide a new perspective for exploring Sarbecovirus' evolution.

Unwinding mechanism of SARS-CoV helicase (nsp13) in the presence of Ca2+, elucidated by biochemical and single-molecular studies.

The recent outbreak of COVID-19 has created a serious health crisis with fatFal infectious viral diseases, such as Severe Acute Respiratory Syndrome (SARS). The nsp13, a helicase of coronaviruses is an essential element for viral replication that unwinds secondary structures of DNA and RNA, and is thus considered a major therapeutic target for treatment. The replication of coronaviruses and other retroviruses occurs in the cytoplasm of infected cells, in association with viral replication organelles, called virus-induced cytosolic double-membrane vesicles (DMVs). In addition, an increase in cytosolic Ca2+ concentration accelerates viral replication. However, the molecular mechanism of nsp13 in the presence of Ca2+ is not well understood. In this study, we applied biochemical methods and single-molecule techniques to demonstrate how nsp13 achieves its unwinding activity while performing ATP hydrolysis in the presence of Ca2+. Our study found that nsp13 could efficiently unwind double stranded (ds) DNA under physiological concentration of Ca2+ of cytosolic DMVs. These findings provide new insights into the properties of nsp13 in the range of calcium in cytosolic DMVs.

hACE2-Induced Allosteric Activation in SARS-CoV versus SARS-CoV-2 Spike Assemblies Revealed by Structural Dynamics.

SARS-CoV and SARS-CoV-2 cell entry begins when spike glycoprotein (S) docks with the human ACE2 (hACE2) receptor. While the two coronaviruses share a common receptor and architecture of S, they exhibit differences in interactions with hACE2 as well as differences in proteolytic processing of S that trigger the fusion machine. Understanding how those differences impact S activation is key to understand its function and viral pathogenesis. Here, we investigate hACE2-induced activation in SARS-CoV and SARS-CoV-2 S using hydrogen/deuterium-exchange mass spectrometry (HDX-MS). HDX-MS revealed differences in dynamics in unbound S, including open/closed conformational switching and D614G-induced S stability. Upon hACE2 binding, notable differences in transduction of allosteric changes were observed extending from the receptor binding domain to regions proximal to proteolytic cleavage sites and the fusion peptide. Furthermore, we report that dimeric hACE2, the native oligomeric form of the receptor, does not lead to any more pronounced structural effect in S compared to saturated monomeric hACE2 binding. These experiments provide mechanistic insights into receptor-induced activation of Sarbecovirus spike proteins.

Pan-sarbecovirus prophylaxis with human anti-ACE2 monoclonal antibodies.

Human monoclonal antibodies (mAbs) that target the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike protein have been isolated from convalescent individuals and developed into therapeutics for SARS-CoV-2 infection. However, therapeutic mAbs for SARS-CoV-2 have been rendered obsolete by the emergence of mAb-resistant virus variants. Here we report the generation of a set of six human mAbs that bind the human angiotensin-converting enzyme-2 (hACE2) receptor, rather than the SARS-CoV-2 spike protein. We show that these antibodies block infection by all hACE2 binding sarbecoviruses tested, including SARS-CoV-2 ancestral, Delta and Omicron variants at concentrations of ~7-100 ng ml-1. These antibodies target an hACE2 epitope that binds to the SARS-CoV-2 spike, but they do not inhibit hACE2 enzymatic activity nor do they induce cell-surface depletion of hACE2. They have favourable pharmacology, protect hACE2 knock-in mice against SARS-CoV-2 infection and should present a high genetic barrier to the acquisition of resistance. These antibodies should be useful prophylactic and treatment agents against any current or future SARS-CoV-2 variants and might be useful to treat infection with any hACE2-binding sarbecoviruses that emerge in the future.

Broadly neutralizing antibodies against sarbecoviruses generated by immunization of macaques with an AS03-adjuvanted COVID-19 vaccine.

The rapid emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants that evade immunity elicited by vaccination has placed an imperative on the development of countermeasures that provide broad protection against SARS-CoV-2 and related sarbecoviruses. Here, we identified extremely potent monoclonal antibodies (mAbs) that neutralized multiple sarbecoviruses from macaques vaccinated with AS03-adjuvanted monovalent subunit vaccines. Longitudinal analysis revealed progressive accumulation of somatic mutation in the immunoglobulin genes of antigen-specific memory B cells (MBCs) for at least 1 year after primary vaccination. Antibodies generated from these antigen-specific MBCs at 5 to 12 months after vaccination displayed greater potency and breadth relative to those identified at 1.4 months. Fifteen of the 338 (about 4.4%) antibodies isolated at 1.4 to 6 months after the primary vaccination showed potency against SARS-CoV-2 BA.1, despite the absence of serum BA.1 neutralization. 25F9 and 20A7 neutralized authentic clade 1 sarbecoviruses (SARS-CoV, WIV-1, SHC014, SARS-CoV-2 D614G, BA.1, and Pangolin-GD) and vesicular stomatitis virus-pseudotyped clade 3 sarbecoviruses (BtKY72 and PRD-0038). 20A7 and 27A12 showed potent neutralization against all SARS-CoV-2 variants and multiple Omicron sublineages, including BA.1, BA.2, BA.3, BA.4/5, BQ.1, BQ.1.1, and XBB. Crystallography studies revealed the molecular basis of broad and potent neutralization through targeting conserved sites within the RBD. Prophylactic protection of 25F9, 20A7, and 27A12 was confirmed in mice, and administration of 25F9 particularly provided complete protection against SARS-CoV-2, BA.1, SARS-CoV, and SHC014 challenge. These data underscore the extremely potent and broad activity of these mAbs against sarbecoviruses.

Environmental risk caused by drug waste in the city of Rio de Janeiro, Brazil, during the SARS-Cov19 pandemic. / Risco ambiental provocado por resíduos de medicamentos na cidade do Rio de Janeiro, Brasil, durante a pandemia por SARS-Cov19.

The relationship between the distribution of medicines used in the Pandemic by SARS-COV-19 in the municipality of Rio de Janeiro and the estimated level of environmental risk caused by their residues was evaluated. The amount of medicines distributed by primary health care (PHC) units between 2019 and 2021 were collected. The risk quotient (RQ) corresponded to the ratio between the estimated predictive environmental concentration (PECest) obtained by the consumption and excretion of each drug and its non-effective predictive concentration (PNEC). Between 2019 and 2020, the PECest of azithromycin (AZI) and ivermectin (IVE) increased between 2019 and 2020, with a decrease in 2021 probably due to shortages. Dexchlorpheniramine (DEX) and fluoxetine (FLU) fell, returning to growth in 2021. While the PECest of diazepam (DIA) increased over these 3 years, ethinylestradiol (EE2) decreased possibly due to the prioritization of PHC in the treatment of COVID-19. The largest QR were from FLU, EE2 and AZI. The consumption pattern of these drugs did not reflect their environmental risk because the most consumed ones have low toxicity. It is worth noting that some data may be underestimated due to the incentive given during the pandemic to the consumption of certain groups of drugs.

Foi avaliada a relação entre a distribuição de medicamentos usados na pandemia por SARS-COV-19 no município do Rio de Janeiro e o nível de risco ambiental estimado provocado por seus resíduos. Foi coletada a quantidade de medicamentos distribuídos pelas unidades de atenção primária à saúde (APS) entre 2019 e 2021. O quociente de risco (QR) correspondeu à razão entre a concentração ambiental preditiva estimada (PECest), obtida pelo consumo e excreção de cada fármaco, e a sua concentração preditiva não efetiva (PNEC). Os PECest da azitromicina e da ivermectina aumentaram entre 2019 e 2020, tendo uma queda em 2021 provavelmente devido ao desabastecimento. Já o da dexclorfeniramina (DEX) e da fluoxetina (FLU) tiveram uma queda, retornando o crescimento em 2021. Enquanto o PECest do diazepam (DIA) aumentou ao longo desses três anos, o etinilestradiol (EE2) diminuiu, possivelmente pela priorização da APS no tratamento da COVID-19. Os maiores QR foram de FLU, EE2 e AZI. O padrão de consumo desses medicamentos não refletiu seu risco ambiental, pois os mais consumidos possuem baixa toxicidade. Vale destacar que alguns dados podem estar subestimados devido ao incentivo que foi dado durante a pandemia para o consumo de determinados grupos de fármacos.