Saliva as a Viable and Simple Alternative to Nasopharyngeal and Oropharyngeal Swabs for COVID-19 Real-Time Reverse Transcriptase Polymerase Chain Reaction

Introduction: Attributable to the difficulties in specimen collection, discomfort and symptoms caused on by Nasopharyngeal (NPS) and Oropharyngeal Swab (OPS) collection, and significant risk to Healthcare Workers (HCW), evaluation of an alternative specimen for the diagnosis of Coronavirus Disease 2019 (COVID-19) is required. Saliva specimen could be an alternative specimen with many advantages over NPS and OPS, however little is known about how well it performs this purpose. Aim(s): The present study was conducted to assess the efficacy of saliva as a viable and simple alternative specimen to NPS and OPS for COVID-19 Real-Time reverse transcriptase Polymerase Chain Reaction (rRT-PCR). Material(s) and Method(s): The present cross-sectional study was conducted in the Department of Microbiology, SGT Medical College Hospital and Research Institute, Haryana, India, from July 2020 to December 2020. A total of 60 symptomatic and 20 asymptomatic COVID-19 patients were recruited for the study and specimen viz., saliva, NPS and OPS were collected at four different sampling points i.e., on day 1, 5, 7 and 14 after confirmation of COVID-19 rRT-PCR test positivity. Data obtained from the study was analysed and expressed as median, frequency, interquartile range and Chi-square test was done for comparison of categorical variables. Result(s): Majority of the patients in symptomatic hospitalised COVID-19 patients were males (n=49, 81.7%) and remaining were females (n=11, 18.3%) and in asymptomatic group 8 (40%) were males and 12 (60%) were females. Saliva was the most sensitive specimen (74.2%), followed by NPS, Naso Oropharyngeal Swab (NOPS) with 70.8% each and OPS (65.8%) for detection of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) in symptomatic patients at four different sampling points. Comparable findings were also observed in specimens obtained from asymptomatic individuals as well. In addition, the viral load was also highest in saliva sample, as measured by Cycle Threshold (CT)-value. Across all specimen types, high viral load (lower CT-values) was observed during the early period of infection. Majority of the study participants reported discomfort during NPS and OPS collection (90% and 85%, respectively), lacrimation, sneezing and gag reflex being the most commonly reported induced symptoms. Conclusion(s): In the present study, saliva could be a viable and alternate specimen for COVID-19 diagnosis due to its ease in sample collection, specimen stability and reduced risk of transmission of infection due to droplets.Copyright © 2023 Journal of Clinical and Diagnostic Research. All rights reserved.

DLin-MC3-Containing mRNA Lipid Nanoparticles Induce an Antibody Th2-Biased Immune Response Polarization in a Delivery Route-Dependent Manner in Mice.

mRNA-based vaccines have made a leap forward since the SARS-CoV-2 pandemic and are currently used to develop anti-infectious therapies. If the selection of a delivery system and an optimized mRNA sequence are two key factors to reach in vivo efficacy, the optimal administration route for those vaccines remains unclear. We investigated the influence of lipid components and immunization route regarding the intensity and quality of humoral immune responses in mice. The immunogenicity of HIV-p55Gag encoded mRNA encapsulated into D-Lin-MC3-DMA or GenVoy-ionizable lipid-based LNPs was compared after intramuscular or subcutaneous routes. Three sequential mRNA vaccines were administrated followed by a heterologous boost composed of p24-HIV protein antigen. Despite equivalent IgG kinetic profiles of general humoral responses, IgG1/IgG2a ratio analysis showed a Th2/Th1 balance toward a Th1-biased cellular immune response when both LNPs were administrated via the intramuscular route. Surprisingly, a Th2-biased antibody immunity was observed when DLin-containing vaccine was injected subcutaneously. A protein-based vaccine boost appeared to reverse this balance to a cellular-biased response correlated to an increase in antibody avidity. Our finding suggests that the intrinsic adjuvant effect of ionizable lipids appears to be dependent on the delivery route used, which could be relevant to reach potent and long-lasting immunity after mRNA-based immunization.

Alterations in heparan sulfate proteoglycan synthesis and sulfation and the impact on vascular endothelial function.

The glycocalyx attached to the apical surface of vascular endothelial cells is a rich network of proteoglycans, glycosaminoglycans, and glycoproteins with instrumental roles in vascular homeostasis. Given their molecular complexity and ability to interact with the intra- and extracellular environment, heparan sulfate proteoglycans uniquely contribute to the glycocalyx's role in regulating endothelial permeability, mechanosignaling, and ligand recognition by cognate cell surface receptors. Much attention has recently been devoted to the enzymatic shedding of heparan sulfate proteoglycans from the endothelial glycocalyx and its impact on vascular function. However, other molecular modifications to heparan sulfate proteoglycans are possible and may have equal or complementary clinical significance. In this narrative review, we focus on putative mechanisms driving non-proteolytic changes in heparan sulfate proteoglycan expression and alterations in the sulfation of heparan sulfate side chains within the endothelial glycocalyx. We then discuss how these specific changes to the endothelial glycocalyx impact endothelial cell function and highlight therapeutic strategies to target or potentially reverse these pathologic changes.

Viral and Host Factors Regulating HIV-1 Envelope Protein Trafficking and Particle Incorporation.

The HIV-1 envelope glycoprotein (Env) is an essential structural component of the virus, serving as the receptor-binding protein and principal neutralizing determinant. Env trimers are incorporated into developing particles at the plasma membrane of infected cells. Incorporation of HIV-1 Env into particles in T cells and macrophages is regulated by the long Env cytoplasmic tail (CT) and the matrix region of Gag. The CT incorporates motifs that interact with cellular factors involved in endosomal trafficking. Env follows an unusual pathway to arrive at the site of particle assembly, first traversing the secretory pathway to the plasma membrane (PM), then undergoing endocytosis, followed by directed sorting to the site of particle assembly on the PM. Many aspects of Env trafficking remain to be defined, including the sequential events that occur following endocytosis, leading to productive recycling and particle incorporation. This review focuses on the host factors and pathways involved in Env trafficking, and discusses leading models of Env incorporation into particles.

ROLE of ACTIN REGULATORS in HIV-1, IAV, & SARS-CoV-2 VIRUS-LIKE PARTICLES PRODUCTION

Background: Human immunodeficiency virus (HIV) and Influenza A virus (IAV) remain a global health concern. Further, emergence of novel coronavirus SARS-CoV-2, which rapidly became global pandemic, increases the concern in biomedical research field for antiviral treatment. To develop new antiviral therapy, we must need to understand the molecular and cellular mechanisms involved in assembly and replication. It is known for some viruses (HIV and IAV) that the host actin cytoskeleton has been involved in various stages of the virus life cycle. Regulation of actin cytoskeleton requires several actin binding proteins, which organize the actin filaments (F-actin) into higher order structures such as actin bundles, branches, filopodia and microvilli, for further assistance in viral particle production. Thus, our objective for this work is to understand the role of these actin regulator proteins, like cofilin and one of its cofactor WDR1, in viral particle assembly and release. Methods: Here we used a combination of different experimental methods like RNA interference, immunoblot, immunoprecipitation, immunofluorescence coupled to confocal and STED fluorescence microscopy. In order to study only virus release, and bypass viral entry, we set up a minimal system for virus-like particles production in transfected cells, giving HIV-1 Gag-VLP, Influenza M1-VLP and SARS-CoV-2 MNE-VLP (developed by D. Muriaux lab). For image analysis, we used Image J software. Statistical analysis was performed with non-parametric t-tests or one-way Anova test. Results: Using siRNA strategy, we have shown that upon knock down of actin protein cofilin or WDR1, HIV-1 and IAV particles production increases in contrario to SARS-CoV-2 VLP release. Further, using immunoprecipitation, we report that HIV-1 Gag is able to form an intracellular complex with WDR1 and cofilin. Similarly, IAV-M1, which like HIV Gag-MA binds with plasma membrane phospholipids, is able to form an intracellular complex with cofilin. These results suggested that virus budding from the host cell plasma membrane seemed restricted by the cofilin/WDR1 complex. Finally, using confocal/STED microscopy on cell producing VLP, we observed actin fibers rearrangement with cell protrusions, suggesting a role for actin in viral particles assembly and release. Conclusion: In conclusion, regulators of actin dynamic are involved in HIV-1 Gag, IAV-M1 and SARS-CoV-2 VLP production but play a differential role in assembly and release of these RNA enveloped viruses.

The endothelial glycocalyx in critical illness: A pediatric perspective.

The vascular endothelium is the interface between circulating blood and end organs and thus has a critical role in preserving organ function. The endothelium is lined by a glycan-rich glycocalyx that uniquely contributes to endothelial function through its regulation of leukocyte and platelet interactions with the vessel wall, vascular permeability, coagulation, and vasoreactivity. Degradation of the endothelial glycocalyx can thus promote vascular dysfunction, inflammation propagation, and organ injury. The endothelial glycocalyx and its role in vascular pathophysiology has gained increasing attention over the last decade. While studies characterizing vascular glycocalyx injury and its downstream consequences in a host of adult human diseases and in animal models has burgeoned, studies evaluating glycocalyx damage in pediatric diseases are relatively few. As children have unique physiology that differs from adults, significant knowledge gaps remain in our understanding of the causes and effects of endothelial glycocalyx disintegrity in pediatric critical illness. In this narrative literature overview, we offer a unique perspective on the role of the endothelial glycocalyx in pediatric critical illness, drawing from adult and preclinical data in addition to pediatric clinical experience to elucidate how marked derangement of the endothelial surface layer may contribute to aberrant vascular biology in children. By calling attention to this nascent field, we hope to increase research efforts to address important knowledge gaps in pediatric vascular biology that may inform the development of novel therapeutic strategies.

A bioorthogonal chemical reporter for fatty acid synthase-dependent protein acylation.

Mammalian cells acquire fatty acids (FAs) from dietary sources or via de novo palmitate production by fatty acid synthase (FASN). Although most cells express FASN at low levels, it is upregulated in cancers of the breast, prostate, and liver, among others, and is required during the replication of many viruses, such as dengue virus, hepatitis C, HIV-1, hepatitis B, and severe acute respiratory syndrome coronavirus 2, among others. The precise role of FASN in disease pathogenesis is poorly understood, and whether de novo FA synthesis contributes to host or viral protein acylation has been traditionally difficult to study. Here, we describe a cell-permeable and click chemistry-compatible alkynyl acetate analog (alkynyl acetic acid or 5-hexynoic acid [Alk-4]) that functions as a reporter of FASN-dependent protein acylation. In an FASN-dependent manner, Alk-4 selectively labels the cellular protein interferon-induced transmembrane protein 3 at its known palmitoylation sites, a process that is essential for the antiviral activity of the protein, and the HIV-1 matrix protein at its known myristoylation site, a process that is required for membrane targeting and particle assembly. Alk-4 metabolic labeling also enabled biotin-based purification and identification of more than 200 FASN-dependent acylated cellular proteins. Thus, Alk-4 is a useful bioorthogonal tool to selectively probe FASN-mediated protein acylation in normal and diseased states.

Influence of Different Glycoproteins and of the Virion Core on SERINC5 Antiviral Activity.

Host plasma membrane protein SERINC5 is incorporated into budding retrovirus particles where it blocks subsequent entry into susceptible target cells. Three structurally unrelated proteins encoded by diverse retroviruses, human immunodeficiency virus type 1 (HIV-1) Nef, equine infectious anemia virus (EIAV) S2, and ecotropic murine leukemia virus (MLV) GlycoGag, disrupt SERINC5 antiviral activity by redirecting SERINC5 from the site of virion assembly on the plasma membrane to an internal RAB7+ endosomal compartment. Pseudotyping retroviruses with particular glycoproteins, e.g., vesicular stomatitis virus glycoprotein (VSV G), renders the infectivity of particles resistant to inhibition by virion-associated SERINC5. To better understand viral determinants for SERINC5-sensitivity, the effect of SERINC5 was assessed using HIV-1, MLV, and Mason-Pfizer monkey virus (M-PMV) virion cores, pseudotyped with glycoproteins from Arenavirus, Coronavirus, Filovirus, Rhabdovirus, Paramyxovirus, and Orthomyxovirus genera. SERINC5 restricted virions pseudotyped with glycoproteins from several retroviruses, an orthomyxovirus, a rhabdovirus, a paramyxovirus, and an arenavirus. Infectivity of particles pseudotyped with HIV-1, amphotropic-MLV (A-MLV), or influenza A virus (IAV) glycoproteins, was decreased by SERINC5, whether the core was provided by HIV-1, MLV, or M-PMV. In contrast, particles pseudotyped with glycoproteins from M-PMV, parainfluenza virus 5 (PIV5), or rabies virus (RABV) were sensitive to SERINC5, but only with particular retroviral cores. Resistance to SERINC5 did not correlate with reduced SERINC5 incorporation into particles, route of viral entry, or absolute infectivity of the pseudotyped virions. These findings indicate that some non-retroviruses may be sensitive to SERINC5 and that, in addition to the viral glycoprotein, the retroviral core influences sensitivity to SERINC5.

Characterization of HIV-1 virus-like particles and determination of Gag stoichiometry for different production platforms.

The importance of developing new vaccine technologies towards versatile platforms that can cope with global virus outbreaks has been evidenced with the most recent severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic. Virus-like particles (VLPs) are a highly immunogenic, safe, and robust approach that can be used to base several vaccine candidates on. Particularly, HIV-1 Gag VLPs is a flexible system comprising a Gag core surrounded by a lipid bilayer that can be modified to present diverse types of membrane proteins or antigens against several diseases, like influenza, dengue, West Nile virus, or human papillomavirus, where it has been proven successful. The size distribution and structural characteristics of produced VLPs vary depending on the cell line used to produce them. In this study, we established an analytical method of characterization for the Gag protein core and clarified the current variability of Gag stoichiometry in HIV-1 VLPs depending on the cell-based production platform, directly determining the number of Gag molecules per VLP in each case. Three Gag peptides have been validated to quantify the number of monomers using parallel reaction monitoring, an accurate and fast, mass-spectrometry-based method that can be used to assess the quality of the produced Gag VLPs regardless of the cell line used. An average of 3617 ± 17 monomers per VLP was obtained for HEK293, substantially varying between platforms, including mammalian and insect cells. This offers a key advantage in quantification and quality control methods to characterize VLP production at a large scale to accelerate new recombinant vaccine production technologies.

Rationale for the Role of Heparin and Related GAG Antithrombotics in COVID-19 Infection.

The SARS-CoV-2 pandemic has focused attention on prevention, restriction and treatment methods that are acceptable worldwide. This means that they should be simple and inexpensive. This review examines the possible role of glycosaminoglycan (GAG) antithrombotics in the treatment of COVID-19. The pathophysiology of this disease reveals a complex interplay between the hemostatic and immune systems that can be readily disrupted by SARS-CoV-2. Some of the GAG antithrombotics also possess immune-modulatory actions and since they are relatively inexpensive they could play an important role in the management of COVID-19 and its complications.