Inactivation of the enveloped virus phi6 with hydrodynamic cavitation.

The COVID -19 pandemic reminded us that we need better contingency plans to prevent the spread of infectious agents and the occurrence of epidemics or pandemics. Although the transmissibility of SARS-CoV-2 in water has not been confirmed, there are studies that have reported on the presence of infectious coronaviruses in water and wastewater samples. Since standard water treatments are not designed to eliminate viruses, it is of utmost importance to explore advanced treatment processes that can improve water treatment and help inactivate viruses when needed. This is the first study to investigate the effects of hydrodynamic cavitation on the inactivation of bacteriophage phi6, an enveloped virus used as a SARS-CoV-2 surrogate in many studies. In two series of experiments with increasing and constant sample temperature, virus reduction of up to 6.3 logs was achieved. Inactivation of phi6 at temperatures of 10 and 20 °C occurs predominantly by the mechanical effect of cavitation and results in a reduction of up to 4.5 logs. At 30 °C, the reduction increases to up to 6 logs, where the temperature-induced increased susceptibility of the viral lipid envelope makes the virus more prone to inactivation. Furthermore, the control experiments without cavitation showed that the increased temperature alone is not sufficient to cause inactivation, but that additional mechanical stress is still required. The RNA degradation results confirmed that virus inactivation was due to the disrupted lipid bilayer and not to RNA damage. Hydrodynamic cavitation, therefore, has the potential to inactivate current and potentially emerging enveloped pathogenic viruses in water at lower, environmentally relevant temperatures.

Mycolactone: A Broad Spectrum Multitarget Antiviral Active in the Picomolar Range for COVID-19 Prevention and Cure.

We have previously shown computationally that Mycolactone (MLN), a toxin produced by Mycobacterium ulcerans, strongly binds to Munc18b and other proteins, presumably blocking degranulation and exocytosis of blood platelets and mast cells. We investigated the effect of MLN on endocytosis using similar approaches, and it bound strongly to the N-terminal of the clathrin protein and a novel SARS-CoV-2 fusion protein. Experimentally, we found 100% inhibition up to 60 nM and 84% average inhibition at 30 nM in SARS-CoV-2 live viral assays. MLN was also 10× more potent than remdesivir and molnupiravir. MLN's toxicity against human alveolar cell line A549, immortalized human fetal renal cell line HEK293, and human hepatoma cell line Huh7.1 were 17.12%, 40.30%, and 36.25%, respectively. The cytotoxicity IC50 breakpoint ratio versus anti-SARS-CoV-2 activity was more than 65-fold. The IC50 values against the alpha, delta, and Omicron variants were all below 0.020 µM, and 134.6 nM of MLN had 100% inhibition in an entry and spread assays. MLN is eclectic in its actions through its binding to Sec61, AT2R, and the novel fusion protein, making it a good drug candidate for treating and preventing COVID-19 and other similarly transmitted enveloped viruses and pathogens.

Enveloped Viruses: Pathogenetic Targets for Cyanobacterial Lectins.

Lectins are a group of highly specific carbohydrate-binding proteins with a wide spectrum of action, involved in the so-called <<first line>> of body defense. These unique biomolecules show high specificity for various mono- and oligosaccharides, primarily for viral and bacterial glycoconjugates. Cyanobacteria lectins are effective against enveloped viruses and are an appealing alternative to existing synthetic drugs. Virtually complete absence of resistance formation in viruses to these compounds is known. The purpose of this review is to analyze, summarize, and discuss the results of experimental studies in vivo and in vitro, illustrating the mechanisms of action and antiviral effects of lectins obtained from cyanobacteria in relation to the most dangerous and socially significant viruses: SARS-Cov-2, HIV, Ebola viruses, influenza, and hepatitis C. In addition, the article outlines some of the challenges that must be overcome in order to obtain effective antiviral drugs in the future.Copyright © Team of Authors, 2022.

Enveloped Viruses: Pathogenetic Targets for Cyanobacterial Lectins.

Lectins are a group of highly specific carbohydrate-binding proteins with a wide spectrum of action, involved in the so-called <<first line>> of body defense. These unique biomolecules show high specificity for various mono- and oligosaccharides, primarily for viral and bacterial glycoconjugates. Cyanobacteria lectins are effective against enveloped viruses and are an appealing alternative to existing synthetic drugs. Virtually complete absence of resistance formation in viruses to these compounds is known. The purpose of this review is to analyze, summarize, and discuss the results of experimental studies in vivo and in vitro, illustrating the mechanisms of action and antiviral effects of lectins obtained from cyanobacteria in relation to the most dangerous and socially significant viruses: SARS-Cov-2, HIV, Ebola viruses, influenza, and hepatitis C. In addition, the article outlines some of the challenges that must be overcome in order to obtain effective antiviral drugs in the future.Copyright © Team of Authors, 2022.

Viral Entry Inhibitors Targeting Six-Helical Bundle Core against Highly Pathogenic Enveloped Viruses with Class I Fusion Proteins.

Type Ⅰ enveloped viruses bind to cell receptors through surface glycoproteins to initiate infection or undergo receptor-mediated endocytosis and initiate membrane fusion in the acidic environment of endocytic compartments, releasing genetic material into the cell. In the process of membrane fusion, envelope protein exposes fusion peptide, followed by an insertion into the cell membrane or endosomal membrane. Further conformational changes ensue in which the type 1 envelope protein forms a typical six-helix bundle structure, shortening the distance between viral and cell membranes so that fusion can occur. Entry inhibitors targeting viral envelope proteins, or host factors, are effective antiviral agents and have been widely studied. Some have been used clinically, such as T20 and Maraviroc for human immunodeficiency virus 1 (HIV-1) or Myrcludex B for hepatitis D virus (HDV). This review focuses on entry inhibitors that target the six-helical bundle core against highly pathogenic enveloped viruses with class I fusion proteins, including retroviruses, coronaviruses, influenza A viruses, paramyxoviruses, and filoviruses.

Efficient Inactivation of Monkeypox Virus by World Health Organization‒Recommended Hand Rub Formulations and Alcohols.

Increasing nonzoonotic human monkeypox virus (MPXV) infections urge reevaluation of inactivation strategies. We demonstrate efficient inactivation of MPXV by 2 World Health Organization‒recommended alcohol-based hand rub solutions. When compared with other (re)emerging enveloped viruses, MPXV displayed the greatest stability. Our results support rigorous adherence to use of alcohol-based disinfectants.

5-(Perylen-3-ylethynyl)uracil as an antiviral scaffold: Potent suppression of enveloped virus reproduction by 3-methyl derivatives in vitro.

Amphipathic nucleoside and non-nucleoside derivatives of pentacyclic aromatic hydrocarbon perylene are known as potent non-cytotoxic broad-spectrum antivirals. Here we report 3-methyl-5-(perylen-3-ylethynyl)-uracil-1-acetic acid and its amides, a new series of compounds based on a 5-(perylen-3-ylethynyl)-uracil scaffold. The compounds demonstrate pronounced in vitro activity against arthropod-borne viruses, namely tick-borne encephalitis virus (TBEV) and yellow fever virus (YFV), in plaque reduction assays with EC50 values below 1.9 and 1.3 nM, respectively, and Chikungunya virus (CHIKV) in cytopathic effect inhibition test with EC50 values below 3.2 µM. The compounds are active against respiratory viruses as well: severe acute respiratory syndrome-related coronavirus 2 (SARS-CoV-2) in cytopathic effect inhibition test and influenza A virus (IAV) in virus titer reduction experiments are inhibited - EC50 values below 51 nM and 2.2 µM, respectively. The activity stems from the presence of a hydrophobic perylene core, and all of the synthesized compounds exhibit comparable 1O2 generation rates. Nonetheless, activity can vary by orders of magnitude depending on the hydrophilic part of the molecule, suggesting a complex mode of action. A time-of-addition experiment and fluorescent imaging indicate that the compounds inhibit viral fusion in a dose-dependent manner. The localization of the compound in the lipid bilayers and visible damage to the viral envelope suggest the membrane as the primary target. Dramatic reduction of antiviral activity with limited irradiation or under treatment with antioxidants further cements the idea of photoinduced ROS-mediated viral envelope damage being the mode of antiviral action.

Physico-Chemical Mechanisms of the Functioning of Membrane-Active Proteins of Enveloped Viruses.

Over the past few years, the attention of the whole world has been riveted to the emergence of new dangerous strains of viruses, among which a special place is occupied by coronaviruses that have overcome the interspecies barrier in the past 20 years: SARS viruses (SARS), Middle East respiratory syndrome (MERS), as well as a new coronavirus infection (SARS-CoV-2), which caused the largest pandemic since the Spanish flu in 1918. Coronaviruses are members of a class of enveloped viruses that have a lipoprotein envelope. This class also includes such serious pathogens as human immunodeficiency virus (HIV), hepatitis, Ebola virus, influenza, etc. Despite significant differences in the clinical picture of the course of disease caused by enveloped viruses, they themselves have a number of characteristic features, which determine their commonality. Regardless of the way of penetration into the cell-by endocytosis or direct fusion with the cell membrane-enveloped viruses are characterized by the following stages of interaction with the target cell: binding to receptors on the cell surface, interaction of the surface glycoproteins of the virus with the membrane structures of the infected cell, fusion of the lipid envelope of the virion with plasma or endosomal membrane, destruction of the protein capsid and its dissociation from the viral nucleoprotein. Subsequently, within the infected cell, the newly synthesized viral proteins must self-assemble on various membrane structures to form a progeny virion. Thus, both the initial stages of viral infection and the assembly and release of new viral particles are associated with the activity of viral proteins in relation to the cell membrane and its organelles. This review is devoted to the analysis of physicochemical mechanisms of functioning of the main structural proteins of a number of enveloped viruses in order to identify possible strategies for the membrane activity of such proteins at various stages of viral infection of the cell.

Mechanics of diffusion-mediated budding and implications for virus replication and infection.

Budding allows virus replication and macromolecular secretion in cells through the formation of a membrane protrusion (bud) that evolves into an envelope. The largest energetic barrier to bud formation is membrane deflection and is trespassed primarily thanks to nucleocapsid-membrane adhesion. Transmembrane proteins (TPs), which later form the virus ligands, are the main promotors of adhesion and can accommodate membrane bending thanks to an induced spontaneous curvature. Adhesive TPs must diffuse across the membrane from remote regions to gather on the bud surface, thus, diffusivity controls the kinetics. This paper proposes a simple model to describe diffusion-mediated budding unravelling important size limitations and size-dependent kinetics. The predicted optimal virion radius, giving the fastest budding, is validated against experiments for coronavirus, HIV, flu and hepatitis. Assuming exponential replication of virions and hereditary size, the model can predict the size distribution of a virus population. This is verified against experiments for SARS-CoV-2. All the above comparisons rely on the premise that budding poses the tightest size constraint. This is true in most cases, as demonstrated in this paper, where the proposed model is extended to describe virus infection via receptor- and clathrin-mediated endocytosis, and via membrane fusion.

Cryo-electron tomography of enveloped viruses.

Viruses are macromolecular machineries that hijack cellular metabolism for replication. Enveloped viruses comprise a large variety of RNA and DNA viruses, many of which are notorious human or animal pathogens. Despite their importance, the presence of lipid bilayers in their assembly has made most enveloped viruses too pleomorphic to be reconstructed as a whole by traditional structural biology methods. Furthermore, structural biology of the viral lifecycle was hindered by the sample thickness. Here, I review the recent advances in the applications of cryo-electron tomography (cryo-ET) on enveloped viral structures and intracellular viral activities.

Virucidal Activity of the Pyridobenzothiazolone Derivative HeE1-17Y against Enveloped RNA Viruses.

Pyridobenzothiazolone derivatives are a promising class of broad-spectrum antivirals. However, the mode of action of these compounds remains poorly understood. The HeE1-17Y derivative has already been shown to be a potent compound against a variety of flaviviruses of global relevance. In this work, the mode of action of HeE1-17Y has been studied for West Nile virus taking advantage of reporter replication particles (RRPs). Viral infectivity was drastically reduced by incubating the compound with the virus before infection, thus suggesting a direct interaction with the viral particles. Indeed, RRPs incubated with the inhibitor appeared to be severely compromised in electron microscopy analysis. HeE1-17Y is active against other enveloped viruses, including SARS-CoV-2, but not against two non-enveloped viruses, suggesting a virucidal mechanism that involves the alteration of the viral membrane.

Direct Lysis RT-qPCR of SARS-CoV-2 in Cell Culture Supernatant Allows for Fast and Accurate Quantification.

Studying the entire virus replication cycle of SARS-CoV-2 is essential to identify the host factors involved and treatments to combat infection. Quantification of released virions often requires lengthy procedures, whereas quantification of viral RNA in supernatant is faster and applicable to clinical isolates. Viral RNA purification is expensive in terms of time and resources, and is often unsuitable for high-throughput screening. Direct lysis protocols were explored for patient swab samples, but the lack of virus inactivation, cost, sensitivity, and accuracy is hampering their application and usefulness for in vitro studies. Here, we show a highly sensitive, accurate, fast, and cheap direct lysis RT-qPCR method for quantification of SARS-CoV-2 in culture supernatant. This method inactivates the virus and permits detection limits of 0.043 TCID50 virus and <1.89 copy RNA template per reaction. Comparing direct lysis with RNA extraction, a mean difference of +0.69 ± 0.56 cycles was observed. Application of the method to established qPCR methods for RSV (-ve RNA), IAV (segmented -ve RNA), and BHV (dsDNA) showed wider applicability to other enveloped viruses, whereby IAV showed poorer sensitivity. This shows that accurate quantification of SARS-CoV-2 and other enveloped viruses can be achieved using direct lysis protocols, facilitating a wide range of high- and low-throughput applications.

Detection of the Omicron (B.1.1.529) variant of SARS-CoV-2 in aircraft wastewater.

On the 26th of November 2021, the World Health Organization (WHO) designated the newly detected B.1.1.529 lineage of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) the Omicron Variant of Concern (VOC). The genome of the Omicron VOC contains more than 50 mutations, many of which have been associated with increased transmissibility, differing disease severity, and potential to evade immune responses developed for previous VOCs such as Alpha and Delta. In the days since the designation of B.1.1.529 as a VOC, infections with the lineage have been reported in countries around the globe and many countries have implemented travel restrictions and increased border controls in response. We putatively detected the Omicron variant in an aircraft wastewater sample from a flight arriving to Darwin, Australia from Johannesburg, South Africa on the 25th of November 2021 via positive results on the CDC N1, CDC N2, and del(69-70) RT-qPCR assays per guidance from the WHO. The Australian Northern Territory Health Department detected one passenger onboard the flight who was infected with SARS-CoV-2, which was determined to be the Omicron VOC by sequencing of a nasopharyngeal swab sample. Subsequent sequencing of the aircraft wastewater sample using the ARTIC V3 protocol with Nanopore and ATOPlex confirmed the presence of the Omicron variant with a consensus genome that clustered with the B.1.1.529 BA.1 sub-lineage. Our detection and confirmation of a single onboard Omicron infection via aircraft wastewater further bolsters the important role that aircraft wastewater can play as an independent and unintrusive surveillance point for infectious diseases, particularly coronavirus disease 2019.

Non-Woven Infection Prevention Fabrics Coated with Biobased Cranberry Extracts Inactivate Enveloped Viruses Such as SARS-CoV-2 and Multidrug-Resistant Bacteria.

The Coronavirus Disease (COVID-19) pandemic is demanding the rapid action of the authorities and scientific community in order to find new antimicrobial solutions that could inactivate the pathogen SARS-CoV-2 that causes this disease. Gram-positive bacteria contribute to severe pneumonia associated with COVID-19, and their resistance to antibiotics is exponentially increasing. In this regard, non-woven fabrics are currently used for the fabrication of infection prevention clothing such as face masks, caps, scrubs, shirts, trousers, disposable gowns, overalls, hoods, aprons and shoe covers as protective tools against viral and bacterial infections. However, these non-woven fabrics are made of materials that do not exhibit intrinsic antimicrobial activity. Thus, we have here developed non-woven fabrics with antimicrobial coatings of cranberry extracts capable of inactivating enveloped viruses such as SARS-CoV-2 and the bacteriophage phi 6 (about 99% of viral inactivation in 1 min of viral contact), and two multidrug-resistant bacteria: the methicillin-resistant Staphylococcus aureus and the methicillin-resistant Staphylococcus epidermidis. The morphology, thermal and mechanical properties of the produced filters were characterized by optical and electron microscopy, differential scanning calorimetry, thermogravimetry and dynamic mechanical thermal analysis. The non-toxicity of these advanced technologies was ensured using a Caenorhabditis elegans in vivo model. These results open up a new prevention path using natural and biodegradable compounds for the fabrication of infection prevention clothing in the current COVID-19 pandemic and microbial resistant era.

Computational methods to study enveloped viral entry.

The protein-membrane interactions that mediate viral infection occur via loosely ordered, transient assemblies, creating challenges for high-resolution structure determination. Computational methods and in particular molecular dynamics simulation have thus become important adjuncts for integrating experimental data, developing mechanistic models, and suggesting testable hypotheses regarding viral function. However, the large molecular scales of virus-host interaction also create challenges for detailed molecular simulation. For this reason, continuum membrane models have played a large historical role, although they have become less favored for high-resolution models of protein assemblies and lipid organization. Here, we review recent progress in the field, with an emphasis on the insight that has been gained using a mixture of coarse-grained and atomic-resolution molecular dynamics simulations. Based on successes and challenges to date, we suggest a multiresolution strategy that should yield the best mixture of computational efficiency and physical fidelity. This strategy may facilitate further simulations of viral entry by a broader range of viruses, helping illuminate the diversity of viral entry strategies and the essential common elements that can be targeted for antiviral therapies.

Systematic Review and Meta-Analysis of the Persistence of Enveloped Viruses in Environmental Waters and Wastewater in the Absence of Disinfectants.

Enveloped viruses are characterized by a lipid-containing envelope that encapsulates the virion, and they have been the cause of major outbreaks and pandemics. Some enveloped viruses are excreted in feces and other bodily fluids of infected people and animals, raising the question of their fate in the aquatic environment. Consequently, we conducted a systematic review and meta-analysis of the decay rate constants (k) of enveloped viruses from 12 families (i.e., Coronaviridae, Cystoviridae (specifically Phi6), Filoviridae, Hepadnaviridae, Herpesviridae, Orthomyxoviridae, Paramyxoviridae, Pneumoviridae, Poxviridae, Retroviridae, Rhabdoviridae, Togaviridae) in environmental waters and wastewater to evaluate their decay kinetics and identify the environmental and virus characteristics that influence k. A total of 812 k that met inclusion criteria were identified in the literature, with the number of k for each family ranging from 0 to 560, and the virus family averaged values of k ranging from 0.11 d-1 and 1.85 d-1. Virus type (i.e., genus, species, subspecies, or subtype), method of virus enumeration (i.e., culture-based or (RT-)QPCR), and experimental water matrix type, temperature and sterility were found to have significant effects on k. Additionally, enveloped viruses were found to have statistically significantly greater k than nonenveloped viruses. Multiple linear regression models that allow prediction of log10k as a function of virus type, enumeration method, water temperature, and water type are provided for six virus families that had sufficient data available for model fitting (i.e., Coronaviridae, Phi6, Herpesviridae, Orthomyxoviridae, Rhabdoviridae, Togaviridae). Compiled log10k and multiple regression models can be used to inform management of human and animal waste, operation of water and wastewater facilities, and exposure risks to treatment plant workers and communities living in regions that lack treatment facilities. Given limited data available for some enveloped virus families with a potential water-related transmission route, there is need for additional data collection to aid academic researchers, public health agencies, and water and wastewater professionals involved in outbreak response.

Wastewater surveillance demonstrates high predictive value for COVID-19 infection on board repatriation flights to Australia.

Controlling importation and transmission of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) from overseas travelers is essential for countries, such as Australia, New Zealand, and other island nations, that have adopted a suppression strategy to manage very low community transmission. Wastewater surveillance of SARS-CoV-2 RNA has emerged as a promising tool employed in public health response in many countries globally. This study aimed to establish whether the surveillance of aircraft wastewater can be used to provide an additional layer of information to augment individual clinical testing. Wastewater from 37 long-haul flights chartered to repatriate Australians was tested for the presence of SARS-CoV-2 RNA. Children 5 years or older on these flights tested negative for coronavirus disease 19 (COVID-19) (deep nasal and oropharyngeal reverse-transcription (RT)-PCR swab) 48 h before departure. All passengers underwent mandatory quarantine for 14-day post arrival in Howard Springs, NT, Australia. Wastewater from 24 (64.9 %) of the 37 flights tested positive for SARS-CoV-2 RNA. During the 14 day mandatory quarantine, clinical testing identified 112 cases of COVID-19. Surveillance for SARS-CoV-2 RNA in repatriation flight wastewater using pooled results from three RT-qPCR assays demonstrated a positive predictive value (PPV) of 87.5 %, a negative predictive value (NPV) of 76.9 % and 83.7% accuracy for COVID-19 cases during the post-arrival 14-day quarantine period. The study successfully demonstrates that the surveillance of wastewater from aircraft for SARS-CoV-2 can provide an additional and effective tool for informing the management of returning overseas travelers and for monitoring the importation of SARS CoV-2 and other clinically significant pathogens.

Soap, water, and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2): an ancient handwashing strategy for preventing dissemination of a novel virus.

Public Health Agencies worldwide (World Health Organization, United States Centers for Disease Prevention & Control, Chinese Center for Disease Control and Prevention, European Centre for Disease Prevention and Control, etc.) are recommending hand washing with soap and water for preventing the dissemination of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infections. In this review, we have discussed the mechanisms of decontamination by soap and water (involving both removal and inactivation), described the contribution of the various components of formulated soaps to performance as cleansers and to pathogen inactivation, explained why adherence to recommended contact times is critical, evaluated the possible contribution of water temperature to inactivation, discussed the advantages of antimicrobial soaps vs. basic soaps, discussed the differences between use of soap and water vs. alcohol-based hand sanitizers for hand decontamination, and evaluated the limitations and advantages of different methods of drying hands following washing. While the paper emphasizes data applicable to SARS-CoV-2, the topics discussed are germane to most emerging and re-emerging enveloped and non-enveloped viruses and many other pathogen types.

A proposed role for the SARS-CoV-2 nucleocapsid protein in the formation and regulation of biomolecular condensates.

To date, the recently discovered SARS-CoV-2 virus has afflicted >6.9 million people worldwide and disrupted the global economy. Development of effective vaccines or treatments for SARS-CoV-2 infection will be aided by a molecular-level understanding of SARS-CoV-2 proteins and their interactions with host cell proteins. The SARS-CoV-2 nucleocapsid (N) protein is highly homologous to the N protein of SARS-CoV, which is essential for viral RNA replication and packaging into new virions. Emerging models indicate that nucleocapsid proteins of other viruses can form biomolecular condensates to spatiotemporally regulate N protein localization and function. Our bioinformatic analyses, in combination with pre-existing experimental evidence, suggest that the SARS-CoV-2 N protein is capable of forming or regulating biomolecular condensates in vivo by interaction with RNA and key host cell proteins. We discuss multiple models, whereby the N protein of SARS-CoV-2 may harness this activity to regulate viral life cycle and host cell response to viral infection.

Strong alkaline electrolyzed water efficiently inactivates SARS-CoV-2, other viruses, and Gram-negative bacteria.

Air spaces and material surfaces in a pathogen-contaminated environment can often be a source of infection to humans, and disinfection has become a common intervention focused on reducing the contamination levels. In this study, we examined the efficacy of SAIW, a unique electrolyzed water with chlorine-free, high pH, high concentration of dissolved hydrogen, and low oxygen reduction potential, for the inactivation of several viruses and bacteria. Infectivity assays revealed that initial viral titers of enveloped and non-enveloped viruses, including severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), influenza A virus, herpes simplex virus type 1, human coronavirus, feline calicivirus, and canine parvovirus, were reduced by 2.9- to 5.5-log10 within 30 s of SAIW exposure. Similarly, the culturability of three Gram-negative bacteria (Escherichia coli, Salmonella, and Legionella) dropped down by 1.9- to 4.9-log10 within 30 s of SAIW treatment. Mechanistically, treatment with SAIW was found to significantly decrease the binding and subsequent entry efficiencies of SARS-CoV-2 on Vero cells. Finally, we showed that this chlorine-free electrolytic ion water had no acute inhalation toxicity in mice, demonstrating that SAIW holds promise for a safer antiviral and antibacterial disinfectant.