A novel antiviral formulation containing caprylic acid inhibits SARS-CoV-2 infection of a human bronchial epithelial cell model.

A novel proprietary formulation, ViruSAL, has previously been demonstrated to inhibit diverse enveloped viral infections in vitro and in vivo. We evaluated the ability of ViruSAL to inhibit SARS-CoV-2 (severe acute respiratory syndrome coronavirus-2) infectivity, using physiologically relevant models of the human bronchial epithelium, to model early infection of the upper respiratory tract. ViruSAL potently inhibited SARS-CoV-2 infection of human bronchial epithelial cells cultured as an air-liquid interface (ALI) model, in a concentration- and time-dependent manner. Viral infection was completely inhibited when ViruSAL was added to bronchial airway models prior to infection. Importantly, ViruSAL also inhibited viral infection when added to ALI models post-infection. No evidence of cellular toxicity was detected in ViruSAL-treated cells at concentrations that completely abrogated viral infectivity. Moreover, intranasal instillation of ViruSAL to a rat model did not result in any toxicity or pathological changes. Together these findings highlight the potential for ViruSAL as a novel and potent antiviral for use within clinical and prophylactic settings.

Air pollution particulate matter as a potential carrier of SARS-CoV-2 to the nervous system and/or neurological symptom enhancer: arguments in favor.

Entry receptor for SARS-CoV-2 is expressed in nasal epithelial cells, and nasal delivery pathway can be a key feature of transmission. Here, a possibility of interaction of SARS-CoV-2 with air pollution particulate matter (PM) was considered. It was shown in our recent studies that water-suspended plastic and wood smoke aerosol PM and carbon-containing nanoparticles from burning organics can interact with the plasma membrane of brain nerve terminals presumably due to their lipid components. COVID-19 patients have neurological symptoms, viral particles were found in the brain, SARS-CoV-2 enters the cells via fusion of lipid viral envelope with the plasma membranes of infected cells, and so viral envelop can contain lipid components of the host neuronal membranes. Therefore, interaction of SARS-CoV-2 envelope with PM is possible in water surrounding. After drying, PM can serve as a carrier for transmission of SARS-CoV-2 immobilized at their surface. Moreover, PM and SARS-CoV-2 per se can enter human organism during nasal inhalation, and they both use the same nose-to-brain delivery pathways moving along axons directly to the brain, influencing the nervous system and exocytosis/endocytosis in nerve cells. Thus, PM can aggravate neurological symptoms of SARS-CoV-2 and vice versa, due to their identical nose-to-brain delivery mechanism and possible interference of neuronal effects. In addition, different types of PM because of their ability to interact with the plasma membranes of nerve cells can facilitate unspecific SARS-CoV-2 entrance to the cells, and can influence envelope features of SARS-CoV-2. Detailed studies are required to analyze interaction of SARS-CoV-2 with PM.

Single-particle detection of native SARS-CoV-2 virions by microfluidic resistive pulse sensing.

Microfluidic resistive pulse sensing (MRPS) was used to determine the size -distribution of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) based on detecting nearly 30,000 single virions. However, the ultrastructure of SARS-CoV-2 is thoroughly described, but ensemble properties of SARS-CoV-2, e.g., its particle size distribution, are sparsely reported. According to the MRPS results, the size distribution of SARS-CoV-2 follows a log-normal function with a mean value of 85.1 nm, which corresponds to an approximate diameter of the viral envelope. This result also confirms the low number (< 50) of spike proteins on the surface of the virions.

Coronaviridae and Tobaniviridae Innovations and Challenges in Electroanalytical Tools for Rapid Biosurveillance of SARS-CoV-2

Summary The International Committee on Taxonomy of Viruses first established the order Nidovirales in 1996. Initially, the order contained only two viral families, Coronaviridae and Arteriviridae . Since the emergence of the human severe acute respiratory syndrome-related coronavirus (SARS-CoV) responsible for the severe acute respiratory syndrome (SARS) in the spring of 2003, coronaviruses became more recognized and generated significant interest among researchers. Coronaviruses are spherical, enveloped virions with large club-shaped surface projections (peplomers) extending from the viral envelope. SARS-CoV-2 disease in hamsters is associated with high levels of virus replication and some of the histopathological changes similar to COVID-19 in humans (e.g., ground-glass opacities). ToVs have recently been classified into the new subfamily Torovirinae within the family Tobaniviridae in the suborder Tornidovirineae . ToVs are associated with enteric diseases in animals and humans, and they are recognized as pathogens of veterinary and medical importance. Since the onset of the coronavirus disease 2019 (COVID-19) pandemic, preventive social paradigms and vaccine development have undergone serious renovations, which drastically reduced the viral spread and increased collective immunity. Although the technological advancements in diagnostic systems for severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) detection are groundbreaking, the lack of sensitive, robust, and consumer-end point-of-care (POC) devices with smartphone connectivity are conspicuously felt. Despite its revolutionary impact on biotechnology and molecular diagnostics, the reverse transcription polymerase chain reaction technique as the gold standard in COVID-19 diagnosis is not suitable for rapid testing. Today's POC tests are dominated by the lateral flow assay technique, with inadequate sensitivity and lack of internet connectivity. Herein, the biosensing advancements in Internet of Things (IoT)-integrated electroanalytical tools as superior POC devices for SARS-CoV-2 detection will be demonstrated. Meanwhile, the impeding factors pivotal for the successful deployment of such novel bioanalytical devices, including the incongruous standards, redundant guidelines, and the limitations of IoT modules will be discussed.

Evidence for SARS-CoV-2 Spike Protein in the Urine of COVID-19 Patients.

Background: SARS-CoV-2 infection has, as of April 2021, affected >133 million people worldwide, causing >2.5 million deaths. Because the large majority of individuals infected with SARS-CoV-2 are asymptomatic, major concerns have been raised about possible long-term consequences of the infection. Methods: Wedeveloped an antigen capture assay to detect SARS-CoV-2 spike protein in urine samples from patients with COVID-19whose diagnosis was confirmed by positive PCR results from nasopharyngeal swabs (NP-PCR+) forSARS-CoV-2. We used a collection of 233 urine samples from 132 participants from Yale New Haven Hospital and the Children's Hospital of Philadelphia that were obtained during the pandemic (106 NP-PCR+ and 26 NP-PCR-), and a collection of 20 urine samples from 20 individuals collected before the pandemic. Results: Our analysis identified 23 out of 91 (25%) NP-PCR+ adult participants with SARS-CoV-2 spike S1 protein in urine (Ur-S+). Interestingly, although all NP-PCR+ children were Ur-S-, one child who was NP-PCR- was found to be positive for spike protein in their urine. Of the 23 adults who were Ur-S+, only one individual showed detectable viral RNA in urine. Our analysis further showed that 24% and 21% of adults who were NP-PCR+ had high levels of albumin and cystatin C, respectively, in their urine. Among individuals with albuminuria (>0.3 mg/mg of creatinine), statistical correlation could be found between albumin and spike protein in urine. Conclusions: Together, our data showed that one of four individuals infected with SARS-CoV-2 develop renal abnormalities, such as albuminuria. Awareness about the long-term effect of these findings is warranted.

MARCH8 Targets Cytoplasmic Lysine Residues of Various Viral Envelope Glycoproteins.

The host transmembrane protein MARCH8 is a RING finger E3 ubiquitin ligase that downregulates various host transmembrane proteins, such as MHC-II. We have recently reported that MARCH8 expression in virus-producing cells impairs viral infectivity by reducing virion incorporation of not only HIV-1 envelope glycoprotein but also vesicular stomatitis virus G-glycoprotein through two different pathways. However, the MARCH8 inhibition spectrum remains largely unknown. Here, we show the antiviral spectrum of MARCH8 using viruses pseudotyped with a variety of viral envelope glycoproteins. Infection experiments revealed that viral envelope glycoproteins derived from the rhabdovirus, arenavirus, coronavirus, and togavirus (alphavirus) families were sensitive to MARCH8-mediated inhibition. Lysine mutations at the cytoplasmic tails of rabies virus-G, lymphocytic choriomeningitis virus glycoproteins, SARS-CoV and SARS-CoV-2 spike proteins, and Chikungunya virus and Ross River virus E2 proteins conferred resistance to MARCH8. Immunofluorescence showed impaired downregulation of the mutants of these viral envelope glycoproteins by MARCH8, followed by lysosomal degradation, suggesting that MARCH8-mediated ubiquitination leads to intracellular degradation of these envelopes. Indeed, rabies virus-G and Chikungunya virus E2 proteins proved to be clearly ubiquitinated. We conclude that MARCH8 has inhibitory activity on a variety of viral envelope glycoproteins whose cytoplasmic lysine residues are targeted by this antiviral factor. IMPORTANCE A member of the MARCH E3 ubiquitin ligase family, MARCH8, downregulates many different kinds of host transmembrane proteins, resulting in the regulation of cellular homeostasis. On the other hands, MARCH8 acts as an antiviral factor when it binds to and downregulates HIV-1 envelope glycoprotein and vesicular stomatitis virus G-glycoprotein that are viral transmembrane proteins. This study reveals that, as in the case of cellular membrane proteins, MARCH8 shows broad-spectrum inhibition against various viral envelope glycoproteins by recognizing their cytoplasmic lysine residues, resulting in lysosomal degradation.

Atom counting method for determining elemental composition of viruses and its applications in biothermodynamics and environmental science.

Quantitative physicochemical perspective on life processes has been a great asset, in bioengineering and biotechnology. The quantitative physicochemical approach can be applied to practically all organisms, including viruses, if their chemical composition and thermodynamic properties are known. In this paper, a new method is suggested for determining elemental composition of viruses, based on atom counting. The atom counting method requires knowledge of genetic sequence, protein sequences and protein copy numbers. An algorithm was suggested for a program that finds elemental composition of various viruses (DNA or RNA, enveloped or non-enveloped). Except for the nucleic acid, capsid proteins, lipid bilayer and carbohydrates, this method includes membrane proteins, as well as spike proteins. The atom counting method has been compared with the existing molecular composition and geometric methods on 5 viruses of different morphology, as well as experimentally determined composition of the poliovirus. The atom counting method was found to be more accurate in most cases. The three methods were found to be complementary, since they require different kind of input information. Moreover, since the 3 methods rest on different assumptions, results of one model can be compared to those of the other two.

S-acylation controls SARS-CoV-2 membrane lipid organization and enhances infectivity.

SARS-CoV-2 virions are surrounded by a lipid bilayer that contains membrane proteins such as spike, responsible for target-cell binding and virus fusion. We found that during SARS-CoV-2 infection, spike becomes lipid modified, through the sequential action of the S-acyltransferases ZDHHC20 and 9. Particularly striking is the rapid acylation of spike on 10 cytosolic cysteines within the ER and Golgi. Using a combination of computational, lipidomics, and biochemical approaches, we show that this massive lipidation controls spike biogenesis and degradation, and drives the formation of localized ordered cholesterol and sphingolipid-rich lipid nanodomains in the early Golgi, where viral budding occurs. Finally, S-acylation of spike allows the formation of viruses with enhanced fusion capacity. Our study points toward S-acylating enzymes and lipid biosynthesis enzymes as novel therapeutic anti-viral targets.

MALDI-ToF protein profiling as a potential rapid diagnostic platform for COVID-19.

More than a year after the COVID-19 pandemic was declared, the need still exists for accurate, rapid, inexpensive and non-invasive diagnostic methods that yield high specificity and sensitivity towards the current and newly emerging SARS-CoV-2 strains. Compared to the nasopharyngeal swabs, several studies have established saliva as a more amenable specimen type for early detection of SARS-CoV-2. Considering the limitations and high demand for COVID-19 testing, we employed MALDI-ToF mass spectrometry in the analysis of 60 gargle samples from human donors and compared the resultant spectra against COVID-19 status. Several standards, including isolated human serum immunoglobulins, and controls, such as pre-COVID-19 saliva and heat inactivated SARS-CoV-2 virus, were simultaneously analyzed to provide a relative view of the saliva and viral proteome as they would appear in this workflow. Five potential biomarker peaks were established that demonstrated high concordance with COVID-19 positive individuals. Overall, the agreement of these results with RT-qPCR testing on nasopharyngeal swabs was ≥90% for the studied cohort, which consisted of young and largely asymptomatic student athletes. From a clinical standpoint, the results from this pilot study suggest that MALDI-ToF could be used to develop a relatively rapid and inexpensive COVID-19 assay.

Challenges of SERS technology as a non-nucleic acid or -antigen detection method for SARS-CoV-2 virus and its variants.

The COVID-19 pandemic has caused a significant burden since December 2019 that has negatively impacted the global economy owing to the fact that the SARS-CoV-2 virus is fast-transmitting and highly contagious. Efforts have been taken to minimize the impact through strict screening measures in country borders in order to isolate potential virus carriers. Effective fast-screening methods are thus needed to identify infected individuals. The standard diagnostic methods for screening SARS-CoV-2 virus have always been to perform nucleic acid-based and serological tests. However, with each having drawbacks on producing false results at very early or later stage after symptoms onset, supplementary techniques are needed to back up these tests. Surface-enhanced Raman spectroscopy (SERS) as a detection technique has continuously advanced throughout the years in terms of sensitivity and capability to detect ultralow concentration of analytes ranging from single molecule to pathogens, to present as a highly potential alternative to known sensing methods. SERS technology as a candidate for an alternative and supplementary diagnostic method for the viral envelope of SARS-CoV-2 virus is presented, comparing its pros and cons to the standard methods and what other aspects it could offer that the other methods are not capable of. Factors that contribute to the detection effectivity of SERS is also discussed to show the advantages and limitations of this technique. Despite its promising capabilities, challenges like sources of SARS-CoV-2 virus and its variations, reliable SERS spectra, mass production of SERS-active substrates, and compliance to regulations for wide-scale testing scenario are highlighted.

MARCH8 Inhibits Ebola Virus Glycoprotein, Human Immunodeficiency Virus Type 1 Envelope Glycoprotein, and Avian Influenza Virus H5N1 Hemagglutinin Maturation.

Membrane-associated RING-CH-type 8 (MARCH8) strongly blocks human immunodeficiency virus type 1 (HIV-1) envelope glycoprotein (Env) incorporation into virions by downregulating its cell surface expression, but the mechanism is still unclear. We now report that MARCH8 also blocks the Ebola virus (EBOV) glycoprotein (GP) incorporation via surface downregulation. To understand how these viral fusion proteins are downregulated, we investigated the effects of MARCH8 on EBOV GP maturation and externalization via the conventional secretion pathway. MARCH8 interacted with EBOV GP and furin when detected by immunoprecipitation and retained the GP/furin complex in the Golgi when their location was tracked by a bimolecular fluorescence complementation (BiFC) assay. MARCH8 did not reduce the GP expression or affect the GP modification by high-mannose N-glycans in the endoplasmic reticulum (ER), but it inhibited the formation of complex N-glycans on the GP in the Golgi. Additionally, the GP O-glycosylation and furin-mediated proteolytic cleavage were also inhibited. Moreover, we identified a novel furin cleavage site on EBOV GP and found that only those fully glycosylated GPs were processed by furin and incorporated into virions. Furthermore, the GP shedding and secretion were all blocked by MARCH8. MARCH8 also blocked the furin-mediated cleavage of HIV-1 Env (gp160) and the highly pathogenic avian influenza virus H5N1 hemagglutinin (HA). We conclude that MARCH8 has a very broad antiviral activity by prohibiting different viral fusion proteins from glycosylation and proteolytic cleavage in the Golgi, which inhibits their transport from the Golgi to the plasma membrane and incorporation into virions.IMPORTANCE Enveloped viruses express three classes of fusion proteins that are required for their entry into host cells via mediating virus and cell membrane fusion. Class I fusion proteins are produced from influenza viruses, retroviruses, Ebola viruses, and coronaviruses. They are first synthesized as a type I transmembrane polypeptide precursor that is subsequently glycosylated and oligomerized. Most of these precursors are cleaved en route to the plasma membrane by a cellular protease furin in the late secretory pathway, generating the trimeric N-terminal receptor-binding and C-terminal fusion subunits. Here, we show that a cellular protein, MARCH8, specifically inhibits the furin-mediated cleavage of EBOV GP, HIV-1 Env, and H5N1 HA. Further analyses uncovered that MARCH8 blocked the EBOV GP glycosylation in the Golgi and inhibited its transport from the Golgi to the plasma membrane. Thus, MARCH8 has a very broad antiviral activity by specifically inactivating different viral fusion proteins.