Virus Receptor Research Articles

Mutations within scavenger receptor cysteine-rich (SRCR) protein domain 5 of porcine CD163 involved in infection with porcine reproductive and respiratory syndrome virus (PRRS).

CD163, a macrophage-specific membrane scavenger receptor, serves as a cellular entry receptor for porcine reproductive and respiratory syndrome virus (PRRSV). The removal of scavenger receptor cysteine-rich (SRCR) domain 5 (SRCR5) of CD163 is sufficient to make transfected cells or genetically modified pigs resistant to PRRSV-1 and PRRSV-2 genotypes, and substitution of SRCR5 with SRCR8 from human CD163-like protein (hCD163L1) confers resistance to PRRSV-1 but not PRRSV-2 isolates. However, the specific regions within the SRCR5 polypeptide involved in PRRSV infection remain largely unknown. In this report, we performed mutational studies in order to identify which regions or amino acid sequences in the SRCR5 domain are critical for PRRSV infection. The approach used in this study was to make proline-arginine (PR) insertions along the SRCR5 polypeptide. Constructs were transfected into HEK293T cells, and then evaluated for infection with PRRSV-2 or PRRSV-1. For PRRSV-2, four PR insertions located after amino acids 8 (PR-9), 47 (PR-48), 54 (PR-55), and 99 (PR-100) had the greatest impact on infection. For PRRSV-1, insertions after amino acids 57 (PR-58) and 99 (PR-100) were critical. Computer simulations based on the crystal structure of SRCR5 showed that the mutations that affected infection localized to a similar region on the surface of the 3-D structure. Specifically, we found two surface patches that are essential for PRRSV infection. PR-58 and PR-55, which were separated by only three amino acids, had reciprocal effects on PRRSV-1 and PRRSV-2. Substitution of Glu-58 with Lys-58 reduced PRRSV-1 infection without affecting PRRSV-2, which partially explains the resistance to PRRSV-1 caused by the SRCR5 replacement with the homolog human SRCR8 previously observed. Finally, resistance to infection was observed following the disruption of any of the four conserved disulfide bonds within SRCR5. In summary, the results confirm that there are distinct differences between PRRSV-1 and PRRSV-2 on recognition of CD163; however, all mutations that affect infection locate on a similar region on the same face of SRCR5.

Inadvertent Transfer of Murine VL30 Retrotransposons to CAR-T Cells.

For more than a decade, genetically engineered autologous T-cells have been successfully employed as immunotherapy drugs for patients with incurable blood cancers. The active components in some of these game-changing medicines are autologous T-cells that express viral vector-delivered chimeric antigen receptors (CARs), which specifically target proteins that are preferentially expressed on cancer cells. Some of these therapeutic CAR expressing T-cells (CAR-Ts) are engineered via transduction with γ-retroviral vectors (γ-RVVs) produced in a stable producer cell line that was derived from murine PG13 packaging cells (ATCC CRL-10686). Earlier studies reported on the copackaging of murine virus-like 30S RNA (VL30) genomes with γ-retroviral vectors generated in murine stable packaging cells. In an earlier study, VL30 mRNA was found to enhance the metastatic potential of human melanoma cells. These findings raise biosafety concerns regarding the possibility that therapeutic CAR-Ts have been inadvertently contaminated with potentially oncogenic VL30 retrotransposons. In this study, we demonstrated the presence of infectious VL30 particles in PG13 cell-conditioned media and observed the ability of these particles to deliver transcriptionally active VL30 genomes to human cells. Notably, VL30 genomes packaged by HIV-1-based vector particles transduced naïve human cells in culture. Furthermore, we detected the transfer and expression of VL30 genomes in clinical-grade CAR-T cells generated by transduction with PG13 cell-derived γ-retroviral vectors. Our findings raise biosafety concerns regarding the use of murine packaging cell lines in ongoing clinical applications.

TNF-α/IFN-γ synergy amplifies senescence-associated inflammation and SARS-CoV-2 receptor expression via hyper-activated JAK/STAT1.

Older age and underlying conditions such as diabetes/obesity or immunosuppression are leading host risk factors for developing severe complications from COVID‐19 infection. The pathogenesis of COVID‐19‐related cytokine storm, tissue damage, and fibrosis may be interconnected with fundamental aging processes, including dysregulated immune responses and cellular senescence. Here, we examined effects of key cytokines linked to cellular senescence on expression of SARS‐CoV‐2 viral entry receptors. We found exposure of human umbilical vein endothelial cells (HUVECs) to the inflammatory cytokines, TNF‐α + IFN‐γ or a cocktail of TNF‐α + IFN‐γ + IL‐6, increased expression of ACE2/DPP4, accentuated the pro‐inflammatory senescence‐associated secretory phenotype (SASP), and decreased cellular proliferative capacity, consistent with progression towards a cellular senescence‐like state. IL‐6 by itself failed to induce substantial effects on viral entry receptors or SASP‐related genes, while synergy between TNF‐α and IFN‐γ initiated a positive feedback loop via hyper‐activation of the JAK/STAT1 pathway, causing SASP amplification. Breaking the interactive loop between senescence and cytokine secretion with JAK inhibitor ruxolitinib or antiviral drug remdesivir prevented hyper‐inflammation, normalized SARS‐CoV‐2 entry receptor expression, and restored HUVECs proliferative capacity. This loop appears to underlie cytokine‐mediated viral entry receptor activation and links with senescence and hyper‐inflammation.

A SARS-CoV-2 Spike Receptor Binding Motif Peptide Induces Anti-Spike Antibodies in Mice andIs Recognized by COVID-19 Patients.

The currently devastating pandemic of severe acute respiratory syndrome known as coronavirus disease 2019 or COVID-19 is caused by the coronavirus SARS-CoV-2. Both the virus and the disease have been extensively studied worldwide. A trimeric spike (S) protein expressed on the virus outer bilayer leaflet has been identified as a ligand that allows the virus to penetrate human host cells and cause infection. Its receptor-binding domain (RBD) interacts with the angiotensin-converting enzyme 2 (ACE2), the host-cell viral receptor, and is, therefore, the subject of intense research for the development of virus control means, particularly vaccines. In this work, we search for smaller fragments of the S protein able to elicit virus-neutralizing antibodies, suitable for production by peptide synthesis technology. Based on the analysis of available data, we selected a 72 aa long receptor binding motif (RBM436-507) of RBD. We used ELISA to study the antibody response to each of the three antigens (S protein, its RBD domain and the RBM436-507 synthetic peptide) in humans exposed to the infection and in immunized mice. The seroreactivity analysis showed that anti-RBM antibodies are produced in COVID-19 patients and immunized mice and may exert neutralizing function, although with a frequency lower than anti-S and -RBD. These results provide a basis for further studies towards the development of vaccines or treatments focused on specific regions of the S virus protein, which can benefit from the absence of folding problems, conformational constraints and other advantages of the peptide synthesis production.

Investigation of the Involvement of HHV-6 Encoded Viral Chemokine Receptors in Autoimmune Thyroiditis Development.

ABSTRACTHuman herpesvirus-6 (HHV-6) contains two genes (U12 and U51) that encode putative homologues of human G-protein-coupled receptors like CCR1, CCR3, and CCR5. It has been shown that these viral proteins can be expressed on the surface of epithelial and some peripheral blood mononuclear cells, suggesting that they could potentially induce autoimmunity. We aimed to investigate the possibility of HHV-6 encoded viral chemokine receptors (U12 and U51) involvement in autoimmune thyroiditis (AIT) development by detecting viral peptide specific antibodies in AIT patient samples. Seventy-nine AIT patients whose thyroid tissues were shown to be positive for HHV-6 and 32 blood donors were enrolled in this study. Twenty-eight synthetic peptides derived from HHV-6 U12 and U51 proteins’ amino acid sequences, as well as recombinant human CCR1, CCR3, and CCR5 proteins were used in suspension multiplex immunological assay to detect specific IgG and IgM antibodies. HHV-6 peptide specific IgG and IgM antibodies were found in patients’ samples. AIT patients' samples were found to be more frequently positive for peptide IgGs in comparison to control group’s samples. Even though peptide antibody cross-reactivity with human CCRs was not demonstrated, our results show a new immunogenic HHV-6 antigen—a possible new player in the HHV-6 induced autoimmunity exacerbation.IMPORTANCE The study of human herpesvirus-6 (HHV-6) involvement in autoimmunity development is very challenging, due to the complex nature of this virus. HHV-6 is a ubiquitous, lifelong persistent, and immunomodulating virus, which mainly spreads in solid tissues using cell-to-cell mechanics, and thus can escape from the host’s immune response. It has been implicated as an environmental factor in several autoimmune diseases. An association between HHV-6 and autoimmune thyroiditis has been demonstrated, yet clear mechanism of involvement remains to be elucidated, since the virus can be detected in nearly all autoimmune thyroiditis patient thyroid glands. Our results show new potentially immunogenic human herpesvirus-6 antigens—possible new players in the HHV-6 induced autoimmunity exacerbation, which could be subjects for further research. Together with previously published results, this study described possible mechanisms which may underlie the induction of autoimmune reactivities against thyroid tissues in AIT.

The effect of the probiotic consortia on SARS-CoV-2 infection in ferrets and on human immune cell response in vitro

SummaryProbiotics have been suggested as one solution to counter detrimental health effects by SARS-CoV-2; however, data so far is scarce. We tested the effect of two probiotic consortia, OL-1 and OL-2, against SARS-CoV-2 in ferrets and assessed their effect on cytokine production and transcriptome in a human monocyte-derived macrophage (Mf) and dendritic cell (DC) model. The results showed that the consortia significantly reduced the viral load, modulated immune response, and regulated viral receptor expression in ferrets compared to placebo. In the human Mf and DC model, OL-1 and OL-2-induced cytokine production and genes related to SARS-CoV-2 antiviral immunity. The study results indicate that probiotic stimulation of the ferret immune system leads to improved antiviral immunity against SARS-COV-2, and the genes and cytokines associated with anti-SARS-CoV-2 immunity are stimulated in human immune cells in vitro. The effect of the consortia against SARS-CoV-2 warrants further investigations in human clinical trials.

COVID-19 & differential effects in twins: Insights from Placenta Pathology

IntroductionCOVID-19 has been associated with several adverse pregnancy outcomes, including perinatal loss. Differential effects of COVID-19 in a twin pregnancy may provide unique insights into virus-placental interactions. We present a case of perinatal loss of a female fetus with survival of the male co-twin in a pregnancy complicated by COVID-19 and premature delivery. MethodsViral detection methods recommended by the NICHD task force were used to identify SARS-CoV-2 and its viral receptors in the placentas and fetal tissue (Antoun et al., 2020) [1] ResultsCompared with the surviving twin, we found a more severe intervillous necrosis and a relatively low detection of ACE2 membranous expression in the syncytiotrophoblasts of the female twin that succumbed. DiscussionThe interactions of SARS-CoV-2 and ACE2 at the maternal fetal interface within the placenta may play a significant role in perinatal loss, and the effects of fetal sex and gestational age at time of infection need to be explored further.

The role of platelets in antiviral immunity

The main role of platelets is traditionally assigned to participation in hemostasis reactions. In recent years, the data have appeared on the non-hemostatic platelet-related role and their active participation in inflammatory reactions. These platelet functions are predetermined by their ability to activate and secrete various immunomodulatory cytokines and chemokines. In addition, activated platelets can directly interact with viral receptors. Recently, there has been growing the knowledge regarding platelet-related regulation of diverse cell types. The result of this interaction is, among others, the formation of platelet-leukocyte aggregates, the focusing of neutrophils at the sites of injury, and generation of a scaffold for developing extracellular traps. Thus, platelets are not only participants in coagulation processes, but also important players in the inflammatory process. This lecture details the issues of platelets controlling and modulating host response to viral infection, as well as potential targets for therapeutic intervention.

The assembled and annotated genome of the masked palm civet (Paguma larvata).

BackgroundThe masked palm civet (Paguma larvata) acts as an intermediate host of severe acute respiratory syndrome coronavirus (SARS-CoV), which caused SARS, and transfered this virus from bats to humans. Additionally, P. larvata has the potential to carry a variety of zoonotic viruses that may threaten human health. However, genome resources for P. larvata have not been reported to date.FindingsA chromosome-level genome assembly of P. larvata was generated using PacBio sequencing, Illumina sequencing, and Hi-C technology. The genome assembly was 2.44 Gb in size, of which 95.32% could be grouped into 22 pseudochromosomes, with contig N50 and scaffold N50 values of 12.97 Mb and 111.81 Mb, respectively. A total of 21,582 protein-coding genes were predicted, and 95.20% of the predicted genes were functionally annotated. Phylogenetic analysis of 19 animal species confirmed the close genetic relationship between P. larvata and species belonging to the Felidae family. Gene family clustering revealed 119 unique, 243 significantly expanded, and 58 significantly contracted genes in the P. larvata genome. We identified 971 positively selected genes in P. larvata, and one known human viral receptor gene PDGFRA is positively selected in P. larvata, which is required for human cytomegalovirus infection.ConclusionsThis high-quality genome assembly provides a valuable genomic resource for exploring virus–host interactions. It will also provide a reliable reference for studying the genetic bases of the morphologic characteristics, adaptive evolution, and evolutionary history of this species.

Structural basis of sodium-dependent bile salt uptake into the liver

The liver takes up bile salts from blood to generate bile, enabling absorption of lipophilic nutrients and excretion of metabolites and drugs1. Human Na+–taurocholate co-transporting polypeptide (NTCP) is the main bile salt uptake system in liver. NTCP is also the cellular entry receptor of human hepatitis B and D viruses2,3 (HBV/HDV), and has emerged as an important target for antiviral drugs4. However, the molecular mechanisms underlying NTCP transport and viral receptor functions remain incompletely understood. Here we present cryo-electron microscopy structures of human NTCP in complexes with nanobodies, revealing key conformations of its transport cycle. NTCP undergoes a conformational transition opening a wide transmembrane pore that serves as the transport pathway for bile salts, and exposes key determinant residues for HBV/HDV binding to the outside of the cell. A nanobody that stabilizes pore closure and inward-facing states impairs recognition of the HBV/HDV receptor-binding domain preS1, demonstrating binding selectivity of the viruses for open-to-outside over inward-facing conformations of the NTCP transport cycle. These results provide molecular insights into NTCP ‘gated-pore’ transport and HBV/HDV receptor recognition mechanisms, and are expected to help with development of liver disease therapies targeting NTCP.

Control of CDH1/E-Cadherin Gene Expression and Release of a Soluble Form of E-Cadherin in SARS-CoV-2 Infected Caco-2 Intestinal Cells: Physiopathological Consequences for the Intestinal Forms of COVID-19.

COVID-19 is the biggest pandemic the world has seen this century. Alongside the respiratory damage observed in patients with severe forms of the disease, gastrointestinal symptoms have been frequently reported. These symptoms (e.g., diarrhoea), sometimes precede the development of respiratory tract illnesses, as if the digestive tract was a major target during early SARS-CoV-2 dissemination. We hypothesize that in patients carrying intestinal SARS-CoV-2, the virus may trigger epithelial barrier damage through the disruption of E-cadherin (E-cad) adherens junctions, thereby contributing to the overall gastrointestinal symptoms of COVID-19. Here, we use an intestinal Caco-2 cell line of human origin which expresses the viral receptor/co-receptor as well as the membrane anchored cell surface adhesion protein E-cad to investigate the expression of E-cad after exposure to SARS-CoV-2. We found that the expression of CDH1/E-cad mRNA was significantly lower in cells infected with SARS-CoV-2 at 24 hours post-infection, compared to virus-free Caco-2 cells. The viral receptor ACE2 mRNA expression was specifically down-regulated in SARS-CoV-2-infected Caco-2 cells, while it remained stable in HCoV-OC43-infected Caco-2 cells, a virus which uses HLA class I instead of ACE2 to enter cells. It is worth noting that SARS-CoV-2 induces lower transcription of TMPRSS2 (involved in viral entry) and higher expression of B0AT1 mRNA (that encodes a protein known to co-express with ACE2 on intestinal cells). At 48 hours post-exposure to the virus, we also detected a small but significant increase of soluble E-cad protein (sE-cad) in the culture supernatant of SARS-CoV-2-infected Caco-2 cells. The increase of sE-cad release was also found in the intestinal HT29 cell line when infected by SARS-CoV-2. Beside the dysregulation of E-cad, SARS-CoV-2 infection of Caco-2 cells also leads to the dysregulation of other cell adhesion proteins (occludin, JAMA-A, zonulin, connexin-43 and PECAM-1). Taken together, these results shed light on the fact that infection of Caco-2 cells with SARS-CoV-2 affects tight-, adherens-, and gap-junctions. Moreover, intestinal tissues damage was associated to the intranasal SARS-CoV-2 infection in human ACE2 transgenic mice.

SARS-CoV-2 drives JAK1/2-dependent local complement hyperactivation

Abstract Patients with coronavirus disease 2019 (COVID-19) present a wide range of acute clinical manifestations affecting the lungs, liver, kidneys, and gut. Angiotensin-converting enzyme 2 (ACE2), the best-characterized entry receptor for the disease-causing virus SARS-CoV-2, is highly expressed in the aforementioned tissues. However, the pathways that underlie the disease are still poorly understood. Here, we unexpectedly found that the complement system was one of the intracellular pathways most highly induced by SARS-CoV-2 infection in lung epithelial cells. Infection of respiratory epithelial cells with SARS-CoV-2 generated activated complement component C3a and could be blocked by a cell-permeable inhibitor of complement factor B (CFBi), indicating the presence of an inducible cell-intrinsic C3 convertase in respiratory epithelial cells. Within cells of the bronchoalveolar lavage of patients, distinct signatures of complement activation in myeloid, lymphoid, and epithelial cells tracked with disease severity. Genes induced by SARS-CoV-2 and the drugs that could normalize these genes both implicated the interferon-JAK1/2-STAT1 signaling system and NF-κB as the main drivers of their expression. Ruxolitinib, a JAK1/2 inhibitor, normalized interferon signature genes and all complement gene transcripts induced by SARS-CoV-2 in lung epithelial cell lines but did not affect NF-κB–regulated genes. Ruxolitinib, alone or in combination with the antiviral remdesivir, inhibited C3a protein produced by infected cells. Together, we postulate that combination therapy with JAK inhibitors and drugs that normalize NF-κB signaling could potentially have clinical application for severe COVID-19. This research was financed by the National Heart, Lung, and Blood Institute of the NIH (grant 5K22HL125593 to M. Kazemian; R01HL119215 to J.R.S.); National Institute of General Medical Sciences of the NIH (grant R35GM138283 to M. Kazemian); and Deutsche Forschungsgemeinschaft (fellowship FR3851/2-1 to T. Freiwald) and supported, in part, by the Intramural Research Program of the NIH; the National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK) (project number ZIA/DK075149 to B.A.); the National Heart, Lung, and Blood Institute (NHLBI) (project number ZIA/Hl006223 to C.K.); and the National Institute of Allergy and Infectious Diseases (NIAID) (project number ZIA/AI001175 to M.S.L.). T. Frum is supported by T32DE007057. Funding for part of the work was provided by the University of Michigan Biological Scholars Program (to C.E.W.), LifeARC Charity (to S.K.), and CRUK KHP Centre (to S.K.).

The Effect of Glycosylation Modulators on the Trafficking and Interaction of Spike Protein S1 Subunit and Angiotensin‐Converting Enzyme 2

The betacoronavirus severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2) is the cause of the ongoing worldwide pandemic. The spike (S) glycoprotein of SARS‐CoV‐2 is a heavily glycosylated trimer that binds to host cell receptor angiotensin‐converting enzyme 2 (ACE2), initiating viral entry. Evidence also shows that the S glycoprotein interacts with other receptors including dipeptidyl peptidase IV (DPPIV). Glycosylation is a post‐translational modification important for proper folding and trafficking of membrane and secretory proteins. It plays a critical role not only for host cell proteins, but also in viral virulence, immunity evasion and receptor binding capabilities. This study focused on the modulation of glycosylation and its effects on the trafficking of the S1 subunit, ACE2 and DPPIV, in addition to its effect on their interactions. For this purpose, the S1 subunit was expressed in COS‐1 cells in the presence or absence of N‐butyldeoxynojirimycin (NB‐DNJ), an inhibitor of the ER‐located a‐glucosidases I and II, 1‐deoxymannojirimycin (dMM), an inhibitor of Golgi‐mannosidase I, or benzyl‐N‐acetyl‐α‐galactosaminide (benzyl‐GalNac), an inhibitor of O‐glycosylation. The data demonstrate that NB‐DNJ affects substantially the trafficking and secretion of S1 protein, while its overall synthetic levels remained unchanged. A similar effect was observed upon treatment of the cells with the benzyl‐GalNac. On the contrary, the presence of dMM in the culture medium did not affect the secretion of S1. Together, the data suggest that folding events implicating calnexin in the ER, the site of action of NB‐DNJ, as well as O‐glycans that are modulated by benzyl‐GalNac are crucial for the secretion of the S1 protein. We further investigated the trafficking of ACE2 and DPPIV in lung Calu‐3 and intestinal Caco‐2 cells in the presence or absence of the glycosylation modulators and their interaction with S1. The data demonstrate a reduced binding capacity of S1 to ACE2 in the presence of NB‐DNJ and dMM, while this interaction was enhanced in Calu‐3 cells upon inhibition of O‐glycosylation by benzyl‐GalNac. We conclude that glycosylation modulators differentially act on the secretory pathway of S1 and the receptors. By virtue of its negative impact on both the secretion of S1 as well as interaction with its receptor ACE2, NB‐DNJ may be considered as a potential therapeutic drug that may act at the level of viral entry and exit.

Inhibitory mechanism of Ambroxol and Bromhexine Hydrochlorides as potent blockers of molecular interaction between SARS-CoV-2 spike protein and human angiotensin-converting Enzyme-2

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infects the host cells through interaction of its spike protein with human angiotensin-converting enzyme 2 (hACE-2). High binding affinity between the viral spike protein and host cells hACE-2 receptor has been reported to enhance the viral infection. Thus, the disruption of this molecular interaction will lead to reduction in viral infectivity. This study, therefore, aimed to analyze the inhibitory potentials of two mucolytic drugs; Ambroxol hydrochlorides (AMB) and Bromhexine hydrochlorides (BHH), to serve as potent blockers of these molecular interactions and alters the binding affinity/efficiency between the proteins employing computational techniques. The study examined the effects of binding of each drug at the receptor binding domain (RBD) of the spike protein and the exopeptidase site of hACE-2 on the binding affinity (ΔGbind) and molecular interactions between the two proteins. Binding affinity revealed that the binding of the two drugs at the RBD-ACE-2 site does not alter the binding affinity and molecular interaction between the proteins. However, the binding of AMB (−56.931 kcal/mol) and BHH (−46.354 kcal/mol) at the exopeptidase site of hACE-2, significantly reduced the binding affinities between the proteins compared to the unbound, ACE-2-RBD complex (−64.856 kcal/mol). The result further showed the two compounds have good affinity at the hACE-2 site, inferring they might be potent inhibitors of hACE-2. Residue interaction networks analysis further revealed the binding of the two drugs at the exopeptidase site of hACE-2 reduced the number of interacting amino residues, subsequently leading to loss of interactions between the two proteins, with BHH showing better reduction in the molecular interaction and binding affinity than AMB. The result of the structural analyses additionally, revealed that the binding of the drugs considerably influences the dynamic of the complexes when compared to the unbound complex. The findings from this study suggest the binding of the two drugs at the exopeptidase site reduces the binding effectiveness of the proteins than their binding at the RBD site, and consequently might inhibit viral attachment and entry.

An atlas of human viruses provides new insights into diversity and tissue tropism of human viruses.

Viruses continue to threaten human health. Yet, the complete viral species carried by humans and their infection characteristics have not been fully revealed. This study curated an atlas of human viruses from public databases and literature, and built the Human Virus Database (HVD). The HVD contains 1131 virus species of 54 viral families which were more than twice the number of the human-infecting virus species reported in previous studies. These viruses were identified in human samples including 68 human tissues, the excreta and body fluid. The viral diversity in humans was age-dependent with a peak in the infant and a valley in the teenager. The tissue tropism of viruses was found to be associated with several factors including the viral group (DNA, RNA or reverse-transcribing viruses), enveloped or not, viral genome length and GC content, viral receptors and the virus-interacting proteins. Finally, the tissue tropism of DNA viruses was predicted using a random-forest algorithm with a middle performance. Overall, the study not only provides a valuable resource for further studies of human viruses but also deepens our understanding toward the diversity and tissue tropism of human viruses. The HVD is available at http://computationalbiology.cn/humanVirusBase/#/. Supplementary data are available at Bioinformatics online.

Virucidal activity and mechanism of action of cetylpyridinium chloride against SARS-CoV-2

ObjectiveSevere acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the pathogen causing the coronavirus disease 2019 (COVID-19) global pandemic. Recent studies have shown the importance of the throat and salivary glands as sites of virus replication and transmission. The viral host receptor, angiotensin-converting enzyme 2 (ACE2), is broadly enriched in epithelial cells of the salivary glands and oral mucosae. Oral care products containing cetylpyridinium chloride (CPC) as a bactericidal ingredient are known to exhibit antiviral activity against SARS-CoV-2 in vitro. However, the exact mechanism of action remains unknown.MethodsThis study examined the antiviral activity of CPC against SARS-CoV-2 and its inhibitory effect on the interaction between the viral spike (S) protein and ACE2 using an enzyme-linked immunosorbent assay.ResultsCPC (0.05%, 0.1% and 0.3%) effectively inactivated SARS-CoV-2 within the contact times (20 and 60 s) in directions for use of oral care products in vitro. The binding ability of both the S protein and ACE2 were reduced by CPC.ConclusionsOur results suggest that CPC inhibits the interaction between S protein and ACE2, and thus, reduces infectivity of SARS-CoV-2 and suppresses viral adsorption.

Plasma Membrane-Derived Liposomes Exhibit Robust Antiviral Activity against HSV-1.

Plasma membranes host a plethora of proteins and glycans on their outer surface that are exploited by viruses to enter the cells. In this study, we have utilized this property to limit a viral infection using plasma membrane-derived vesicles. We show that plasma membrane-derived liposomes are prophylactically and therapeutically competent at preventing herpes simplex virus type-1 (HSV-1) infection. Plasma membrane liposomes derived from human corneal epithelial (HCE) cells, which are natural targets of HSV-1 infection, as well as Vero and Chinese hamster ovary (CHO) cells were used in this study. Our study clearly demonstrates that HCE and Vero-derived cellular liposomes, which express the viral entry-specific cell surface protein receptors, exhibit robust antiviral activity especially when compared to CHO-derived liposomes, which lack the relevant HSV-1 entry receptors. Further experimentation of the plasma membrane-derived liposomes with HSV type-2 (HSV-2) and pseudorabies virus yielded similar results, indicating strong potential for the employment of these liposomes to study viral entry mechanisms in a cell free-environment.

Analysis of protein-heparin interactions using a portable SPR instrument

Optical biosensors such as those based on surface plasmon resonance (SPR) are a key analytical tool for understanding biomolecular interactions and function as well as the quantitative analysis of analytes in a wide variety of settings. The advent of portable SPR instruments enables analyses in the field. A critical step in method development is the passivation and functionalisation of the sensor surface. We describe the assembly of a surface of thiolated oleyl ethylene glycol/biotin oleyl ethylene glycol and its functionalisation with streptavidin and reducing end biotinylated heparin for a portable SPR instrument. Such surfaces can be batch prepared and stored. Two examples of the analysis of heparin-binding proteins are presented. The binding of fibroblast growth factor 2 and competition for the binding of a heparan sulfate sulfotransferase by a library of selectively modified heparins and suramin, which identify the selectivity of the enzyme for sulfated structures in the polysaccharide and demonstrate suramin as a competitor for the enzyme’s sugar acceptor site. Heparin functionalised surfaces should have a wide applicability, since this polysaccharide is a close structural analogue of the host cell surface polysaccharide, heparan sulfate, a receptor for many endogenous proteins and viruses.

Synthesis, molecular docking of 3-(2-chloroethyl)-2,6-diphenylpiperidin-4-one: Hirshfeld surface, spectroscopic and DFT based analyses

In this work, 3-(2-chloroethyl)-2,6-diphenylpiperidin-4-one (CEDP) was synthesized, characterized via spectroscopic techniques (FT-IR, FT-Raman, UV–Vis, 1H and 13C NMR) and optimized using the Density Functional Theory (DFT) approach with a hybrid correlation, B3LYP/6–311++G(d,p) level of theory. The molecular electrostatic potential (MEP) and natural bond orbitals (NBO) were calculated. The molecule was also docked at the active sites of Influenza A virus receptors. The NBO results revealed the occurrence of hyperconjugation and delocalization of π-electrons. Hirshfeld surface and fingerprint analysis were used to examine the intermolecular hydrogen bonding and electron density of the crystal structure. The inter contact forces are C···H/H···C (19.9%), O···H/H···O (8.0%), Cl···H/H···Cl (11.8%), H···H (58.7%) and N···H/H···N (0.8%). The energy gap of CEDP was found to be 5.42 eV, suggesting its stability. Extra precision docking results revealed that CEDP binds well with both RNA polymerase PB1-PB2 and neuraminidase receptors, with similar interactions observed as compared with the standard drugs.

Active vitiligo vulgaris following the administration of the Oxford–AstraZeneca (AZD1222) vaccine against SARS-CoV-2

Sir, There is significant uncertainty surrounding vaccines against COVID-19. Due to the critical need, these vaccines are being developed and approved at a uniquely fast pace [1]. Currently, the approved vaccines take their action by administering the host with sequences encoding the viral spike protein. This essentially means that these gene-therapy-based vaccines, whose long-term effects remain unknown, are going to be administered globally [2]. Therefore, despite their safety and efficacy demonstrated in respective clinical trials, it is reasonable to be cautious and adopt more intensive post-marketing vigilance [3]. A forty-nine-year-old diabetic male presented with asymptomatic, rapidly growing, leukodermal patches on the scalp (Fig. 1), which began to appear suddenly fourteen days following the administration of the first dose of the Oxford–AstraZeneca (AZD1222) vaccine against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The patches were clinically consistent with vitiligo and were confirmed with their well-defined, ivory-white patches by Wood’s lamp examination (Fig. 2).There were no leucodermic patches anywhere else on the body, no family history of vitiligo, and no history of COVID-19 viral infection. After one week of reevaluation, the patch increased in size. The patient also presented with cellulitis on the right groin persistent for three days and was treated with an antibiotic with a follow-up. All baseline investigations, including a full blood count and renal and liver function tests, were normal. The patient is currently on follow-up. Cytokines play a role in regulating the immune response and the depigmentation process in vitiligo. There is an imbalance in cytokine levels in patients with vitiligo. IFN-g expression plays a role in the autoimmune process in vitiligo. The expression of the cytokine IFN-g is associated with melanocyte destruction in the active phase of vitiligo lesions [4]. IFN-g interacts with the viral receptor, resulting in the consequent reduction of several virus replicating, down-regulating genes and gene products [5]. Li et al. [6] demonstrated that IFNs are potential drug choices for SARS-CoV-2 infection. Here, we hypothesize that patients with non-segmental vitiligo (NSV), an autoimmune skin (and mucosal) disorder, may clear SARS-CoV-2 infection more efficiently and have a lower risk of COVID-19 development. Conversely, in the case of COVID-19 development, vitiligo autoimmunity may influence the cytokine storm-related disease burden. In addition, immune activation during SARS-CoV-2 infection or COVID-19 disease may increase vitiligo disease activity. Our hypothesis is based on the shift of the immune system in NSV toward adaptive type 1 (IFNg and CD8 T cells) and innate immune responses [7]. It is unclear whether the vitiligo in our patient was caused by vaccination, However, the temporal relationship between the vaccine and the development of the disease is interesting. We may, therefore, conclude that our patient had his latent vitiligo activated by the vaccine. Yet, further work is needed to demonstrate a causal relationship between vitiligo and COVID-19 vaccination. To the best of our knowledge, this is the first reported case of vitiligo associated with the administration of the Oxford–AstraZeneca (AZD1222) vaccine.