Inherited deficiency of the OAS-RNase L pathway in children with MIS-C

Multisystem inflammatory syndrome in children (MIS-C) is a rare and severe condition that follows benign COVID-19. We report autosomal recessive deficiencies of OAS1, OAS2, or RNASEL in five unrelated children with MIS-C. The cytosolic dsRNA-sensing OAS1 and OAS2 generate 2'-5'-linked oligoadenylates (2-5A) that activate the ssRNA-degrading RNase L. Consistent with the absence of pneumonia in these patients, epithelial cells and fibroblasts defective for this pathway restricted SARS-CoV-2 normally. This contrasted with IFNAR1-deficient cells from patients prone to hypoxemic pneumonia without MIS-C. Monocytic cell lines and primary myeloid cells with OAS1, OAS2, or RNASEL deficiencies produce excessive amounts of inflammatory cytokines upon dsRNA or SARS-CoV- 2 stimulation. Exogenous 2-5A suppresses cytokine production in OAS1-but not RNase L- deficient cells. Cytokine production in RNase L-deficient cells is impaired by MDA5 or RIG-I deficiency and abolished by MAVS deficiency. Recessive OAS-RNase L deficiencies in these patients unleash the production of SARS-CoV-2-triggered, MAVS-mediated inflammatory cytokines by mononuclear phagocytes, thereby underlying MIS-C.Copyright © 2023 Elsevier Inc.

Assembling a structural model of the SARS-CoV-2 replication and transcription complex from 60+ subunits

The SARS-CoV-2 replication and transcription complex (RTC) is made up of nine distinct non-structural viral proteins encoded by the ORF1ab gene. These proteins house seven enzymatic sites that synthesize new viral genomic and subgenomic RNA, proofread and correct errors in the synthesis, add a 5'-cap to the nascent RNA, and truncate the intermediate negative sense 5'-poly-U tail. While x-ray crystallography and cryo-EM have provided high resolution structures of each of the individual proteins of the RTC and have shed light on how subsets of the proteins associate, a full picture of the RTC has remained elusive. Using molecular modeling tools, including protein-protein docking, we have generated a model of the RTC centered around hexameric nsp15, which is capped on two faces by trimers of nsp14/nsp16/(nsp10)2. A conformational change of nsp14, necessary to facilitate binding to nsp15, then recruits six nsp12/nsp7/(nsp8)2 polymerase subunits. To this, six nsp13 subunits are distributed around the complex. The resulting superstructure is composed of 60 subunits total and positions the nsp14 exonuclease and nsp15 endonuclease sites in line with the dsRNA exiting the nsp12 polymerase site. Nsp10 acts to separate the RNA strands, directing the nascent strand to the nsp12 NiRAN site, where a transiently associated nsp9 facilitates the first step in mRNA capping. The RNA is then directed to the nsp14 N7-methyltransferase site and the nsp16 2'O-methyltransferase site to complete the capping. Additionally, template switching during transcription is proposed to be facilitated by positioning of the TRS-L RNA-bound N-protein above the polymerase active site, between two subunits of nsp13. The model, while constructed based on structural considerations, offers a unifying set of hypotheses to explain the diverse set of processes involved in coronavirus genome replication and transcription. All work presented was funded by Gilead Sciences.Copyright © 2023 The American Society for Biochemistry and Molecular Biology, Inc.

The SARS-CoV-2 Nsp13 Helicase Required for Coronavirus Replication Interacts with Structured Nucleic Acids in a Strand-Specific Manner and Catalyzes a Novel Protein-RNA Remodeling Activity Implicated in the Proofreading Mechanism of the Replication-Transc

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), a single-stranded, positive-sense RNA virus responsible for COVID-19, requires a set of virally encoded nonstructural proteins that compose a replication-transcription complex (RTC) to replicate its 30 kilobase genome. One such nonstructural protein within the RTC is Nsp13, a highly conserved molecular motor ATPase/helicase. Upon purification of the recombinant SARS-CoV-2 Nsp13 protein expressed using a eukaryotic cell-based system, we biochemically characterized the enzyme by examining its catalytic functions, nucleic acid substrate specificity, and putative protein-nucleic acid remodeling activity. We determined that Nsp13 preferentially interacts with single-stranded (ss) DNA compared to ssRNA during loading to unwind with greater efficiency a partial duplex helicase substrate. The binding affinity of Nsp13 to nucleic acid was confirmed through electrophoretic mobility shift assays (EMSA) by determining that Nsp13 binds to DNA substrates with significantly greater efficiency than RNA. These results demonstrate strand-specific interactions of SARS-CoV-2 Nsp13 that dictate its ability to load and unwind structured nucleic acid substrates. We next determined that Nsp13 catalyzed unwinding of double-stranded (ds) RNA forked duplexes on substrates containing a backbone disruption (neutrally charged polyglycol linker (PGL)) was strongly inhibited when the PGL was positioned in the 5' ssRNA overhang, suggesting an unwinding mechanism in which Nsp13 is strictly sensitive to perturbation of the translocating strand sugar-phosphate backbone integrity. Furthermore, we demonstrated for the first time the ability of the coronavirus Nsp13 helicase to disrupt a high-affinity nucleic acid-protein interaction, i.e., a streptavidin tetramer bound to biotinylated RNA or DNA substrate, in a uni-directional manner and with a preferential displacement of the streptavidin complex from biotinylated ssDNA versus ssRNA. In contrast to the poorly hydrolysable ATP-gamma-S or non-hydrolysable AMP-PNP, ATP supports Nsp13-catalyzed disruption of the nucleic acidprotein complex, suggesting that nucleotide binding by Nsp13 is not sufficient for protein-RNA disruption and the chemical energy of nucleoside triphosphate hydrolysis is required to fuel remodeling of protein bound to RNA or DNA. Our results build upon structural studies of the SARS-CoV-2 RTC in which it was suggested that Nsp13 pushes the RNA polymerase (Nsp12) backward on the template RNA strand. Experimental evidence from our studies demonstrate that Nsp13 helicase efficiently remodels a large high affinity protein-RNA complex in a manner dependent on its intrinsic ATP hydrolysis function. We proposed that this novel biochemical activity of Nsp13 is relevant to its role in SARS-CoV-2 RNA processing functions and replication. It was proposed that Nsp13 facilitates proofreading during coronavirus replication when a mismatched base is inadvertently incorporated into the SARS-CoV-2 genome during replication to reposition the RTC so that the proofreading nuclease complex (Nsp14-Nsp10) can gain access and remove the nascently synthesized nucleotide to ensure polymerase fidelity. Our findings implicate a direct catalytic role of Nsp13 in protein-RNA remodeling during coronavirus genome replication beyond its duplex strand separation or structural stabilization of the RTC, yielding new insight into the proofreading mechanism. This work was supported by the Intramural Training Program, National Institute on Aging (NIA), NIH, and a Special COVID-19 Grant from the Office of the Scientific Director, NIA, NIH.Copyright © 2023 The American Society for Biochemistry and Molecular Biology, Inc.

The Nucleocapsid Proteins of SARS-CoV-2 and Its Close Relative Bat Coronavirus RaTG13 Are Capable of Inhibiting PKR- and RNase L-Mediated Antiviral Pathways.

Coronaviruses (CoVs), including severe acute respiratory syndrome CoV (SARS-CoV), Middle East respiratory syndrome CoV (MERS-CoV), and SARS-CoV-2, produce double-stranded RNA (dsRNA) that activates antiviral pathways such as PKR and OAS/RNase L. To successfully replicate in hosts, viruses must evade such antiviral pathways. Currently, the mechanism of how SARS-CoV-2 antagonizes dsRNA-activated antiviral pathways is unknown. In this study, we demonstrate that the SARS-CoV-2 nucleocapsid (N) protein, the most abundant viral structural protein, is capable of binding to dsRNA and phosphorylated PKR, inhibiting both the PKR and OAS/RNase L pathways. The N protein of the bat coronavirus (bat-CoV) RaTG13, the closest relative of SARS-CoV-2, has a similar ability to inhibit the human PKR and RNase L antiviral pathways. Via mutagenic analysis, we found that the C-terminal domain (CTD) of the N protein is sufficient for binding dsRNA and inhibiting RNase L activity. Interestingly, while the CTD is also sufficient for binding phosphorylated PKR, the inhibition of PKR antiviral activity requires not only the CTD but also the central linker region (LKR). Thus, our findings demonstrate that the SARS-CoV-2 N protein is capable of antagonizing the two critical antiviral pathways activated by viral dsRNA and that its inhibition of PKR activities requires more than dsRNA binding mediated by the CTD. IMPORTANCE The high transmissibility of SARS-CoV-2 is an important viral factor defining the coronavirus disease 2019 (COVID-19) pandemic. To transmit efficiently, SARS-CoV-2 must be capable of disarming the innate immune response of its host efficiently. Here, we describe that the nucleocapsid protein of SARS-CoV-2 is capable of inhibiting two critical innate antiviral pathways, PKR and OAS/RNase L. Moreover, the counterpart of the closest animal coronavirus relative of SARS-CoV-2, bat-CoV RaTG13, can also inhibit human PKR and OAS/RNase L antiviral activities. Thus, the importance of our discovery for understanding the COVID-19 pandemic is 2-fold. First, the ability of SARS-CoV-2 N to inhibit innate antiviral activity is likely a factor contributing to the transmissibility and pathogenicity of the virus. Second, the bat relative of SARS-CoV-2 has the capacity to inhibit human innate immunity, which thus likely contributed to the establishment of infection in humans. The findings described in this study are valuable for developing novel antivirals and vaccines.

Hypothesis of a potential antiviral drug

The approval of mRNA vaccine technique against COVID-19 opens a door to research and the creation of new drugs against different infectious pathologies or even cancer, since for several diseases the therapeutic options are limited, and different viral diseases are treated only symptomatically. For these reasons, this study proposed a hypothesis supported by biological studies, that it provides a theoretical basis for the possible development of a drug that used the mRNA technique and the ribonucleolytic action of a ribonuclease for a possible antiviral therapy, and analyzed a future perspective of this technique in order to provide a bibliographic basis on this hypothesis and motivate researchers to carry out biological studies on this topic.Copyright © 2022, Health Biotechnology and Biopharma. All rights reserved.

IgA vasculitis nephritis (Schonlein-Henoch purpura with nephritis) following COVID-19 vaccination

IgA vasculitis nephritis (Schonlein-Henoch purpura nephritis) is an autoimmune circumstance characterized by palpable purpura involving the lower limbs, arthralgia, abdominal pain and kidney involvement. It is possible that a cytokine storm following coronavirus disease 2019 (COVID-19) could lead to an immunological dysregulation responsible for IgA vasculitis nephritis in these cases. Reactivation or first onset of IgA vasculitis nephritis is uncommon;however, there have been increasing reports of this disease, as a complication of COVID-19 vaccination. It is possible that COVID-19 mRNA vaccination may trigger several auto-inflammatory and autoimmune cascades. Previous research has shown that Toll-like receptors play a role in the development of IgA vasculitis nephritis. Following injection of a COVID-19 mRNA vaccine, the uptake of double-stranded RNA by-products will trigger Toll-like receptors, leading to a series of intracellular cascades starting an innate immunity-driven process of cell-mediated and humoral-mediated immunity.Copyright © 2023 The Author(s);Published by Society of Diabetic Nephropathy Prevention. This is an open-access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Viral enteritis in cattle: To well known viruses and beyond

Livestock products supply about 13 percent of energy and 28 percent of protein in diets consumed worldwide. Diarrhea is a leading cause of sickness and death of beef and dairy calves in their first month of life and also affecting adult cattle, resulting in large economic losses and a negative impact on animal welfare. Despite the usual multifactorial origin, viruses are generally involved, being among the most important causes of diarrhea. There are several viruses that have been confirmed as etiological agents (i.e., rotavirus and coronavirus), and some viruses that are not yet confirmed as etiological agents. This review summarizes the viruses that have been detected in the enteric tract of cattle and tries to deepen and gather knowledge about them.Copyright © 2021 by the authors. Licensee MDPI, Basel, Switzerland.

Immunohistochemical detection of double-stranded RNA in formalin-fixed paraffin-embedded tissue.

Double-stranded RNA (dsRNA) is produced during most viral infections, and immunohistochemical detection of dsRNA has been proposed as a potential screening marker for viral replication. The anti-dsRNA monoclonal antibody clone 9D5 is more sensitive than the established clone J2 but has not been validated in formalin-fixed paraffin-embedded (FFPE) tissue. This study aimed to test and compare the performance of the anti-dsRNA monoclonal antibodies, 9D5 and J2, in FFPE tissue using an automated staining platform. Archived clinical tissue samples with viral infections (n = 34) and uninfected controls (n = 30) were examined. Immunohistochemical staining for dsRNA (9D5 and J2) and virus-specific epitopes was performed. 9D5 provided a similar staining pattern but a higher signal-to-noise ratio than J2. The following proportions of virus-infected tissue samples were dsRNA-positive: SARS-CoV-2 (5/5), HPV (6/6), MCV (5/5), CMV (5/6), HSV (4/6), and EBV (0/6). Also, 18 of 30 uninfected samples were dsRNA positive, and an association between fixation time and intensity was observed. However, signals in all samples were markedly reduced by pretreatment with dsRNA-specific RNAse-III, indicating a specific reaction. In conclusion, dsRNA can be demonstrated in most viral infections with immunohistochemistry in FFPE tissue but with low clinical specificity. The antibody clone 9D5 performs better than clone J2.

Melastoma malabathricum L. Suppresses Neutrophil Extracellular Trap Formation Induced by Synthetic Analog of Viral Double-Stranded RNA Associated with SARS-CoV-2 Infection.

Platelet hyper-reactivity and neutrophil extracellular trap (NET) formation contribute to the development of thromboembolic diseases for patients infected with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). This study investigated the pathophysiological effects of SARS-CoV-2 surface protein components and the viral double-stranded RNA (dsRNA) on platelet aggregation and NET formation. Traditional Chinese medicine (TCM) with anti-viral effects was also delineated. The treatment of human washed platelets with SARS-CoV-2 spike protein S1 or the ectodomain S1 + S2 regions neither caused platelet aggregation nor enhanced agonists-stimulated platelet aggregation. Moreover, NET formation can be induced by polyinosinic-polycytidylic acid (poly(I:C)), a synthetic analog of viral dsRNA, but not by the pseudovirus composed of SARS-CoV-2 spike, envelope, and membrane proteins. To search for TCM with anti-NET activity, the plant Melastoma malabathricum L. which has anticoagulant activity was partially purified by fractionation. One of the fractions inhibited poly(I:C)-induced NET formation in a dose-dependent manner. This study implicates that SARS-CoV-2 structural proteins alone are not sufficient to promote NET and platelet activation. Instead, dsRNA formed during viral replication stimulates NET formation. This study also sheds new insight into using the active components of Melastoma malabathricum L. with anti-NET activity in the battle of thromboembolic diseases associated with SARS-CoV-2 infection.

Double-stranded RNAs are promising adjuvants for enhancing immunogenicity of vaccines

Background. The most effective way to prevent infectious diseases is vaccination. Adjuvants contribute to the optimization of the immune response of vaccines. Double-stranded ribonucleic acids (dsRNAs) from natural sources are promising, but insufficiently studied adjuvants. The aim of the work was to study the adjuvant activity of dsRNA obtained from the killer strain of Saccharomyces cerevisiae using two models of induction of a specific immune response. Materials and methods. In the experiments, the substance of the drug Ridostin containing dsRNA, 21.72% (produced by Institute of Medical Biotechnology of the State Research Center of Virology and Biotechnology "Vector”), was used. A specific immune response was modeled using ovalbumin (OVA) or the substance of the EpiVacCorona vaccine (EVC). The experiments were carried out in 200 female BALB/c mice. Mice of the experimental groups were injected twice with antigen and adjuvant together with a 28-day interval, mice of the comparison group — with antigen only. On the 10th day after the second immunization, blood samples were collected to determine the level of specific antibodies using enzyme immunoassay. The results were evaluated by calculation of the average geometric titers of specific antibodies against OVA or EVC. Results. OVA or EVC administered twice induced the specific antibodies in mice in dose-dependent titers. The combined administration of antigen and dsRNA increased the strength of the immune response. The highest stimulating effect of dsRNA was observed in the dose of 100 µg/mouse administered into mice immunized with OVA (1 µg/mouse) or in the dose of 50 µg/mouse in mice immunized with EVC substance (0.25 of a human dose per mouse). Conclusion. The data obtained indicate that the substance of dsRNA exerts adjuvant properties, which gives reason to consider dsRNA as a promising adjuvant for peptide vaccines. © 2022, Central Research Institute for Epidemiology. All rights reserved.

DEZVOLTAREA UNUI VACCIN ARN MESAGER - CONSIDERENTE GENERALE

Literature review on the technologies available for the development, production and testing of a messenger RNA vaccine, starting with the establishment of the optimal sequence elements, methods for production and purification of nucleic acids and delivery systems used in vaccination and gene therapy studies. The mRNA sequence, especially the 5' and 3' untranslated regulatory elements are essential to increase stability and ensure a high level of antigen expression in vitro and in vivo. Reactogenicity and decrease in antigen expression level due to the activation of in vivo antiviral response mechanisms can be diminished by introducing additional purification steps aimed at eliminating DNA, double-stranded RNA and other contaminants resulting from the in vitro transcription processes.

DEAD-box RNA helicase 21 negatively regulates cytosolic RNA-mediated innate immune signaling.

DEAD-box RNA helicase 21 (DDX21), also known as RHII/Gu, is an ATP-dependent RNA helicase. In addition to playing a vital role in regulating cellular RNA splicing, transcription, and translation, accumulated evidence has suggested that DDX21 is also involved in the regulation of innate immunity. However, whether DDX21 induces or antagonizes type I interferon (IFN-I) production has not been clear and most studies have been performed through ectopic overexpression or RNA interference-mediated knockdown. In this study, we generated DDX21 knockout cell lines and found that knockout of DDX21 enhanced Sendai virus (SeV)-induced IFN-ß production and IFN-stimulated gene (ISG) expression, suggesting that DDX21 is a negative regulator of IFN-ß. Mechanistically, DDX21 competes with retinoic acid-inducible gene I (RIG-I) for binding to double-stranded RNA (dsRNA), thereby attenuating RIG-I-mediated IFN-ß production. We also identified that the 217-784 amino acid region of DDX21 is essential for binding dsRNA and associated with its ability to antagonize IFN production. Taken together, our results clearly demonstrated that DDX21 negatively regulates IFN-ß production and functions to maintain immune homeostasis.

Bovine lactoferrin increases the poly(I:C)-induced antiviral response in vitro.

Bovine lactoferrin (bLF) is a naturally occurring glycoprotein with antibacterial and antiviral activities. We evaluated whether bLF can prevent viral infections in the human intestinal epithelial cell line Caco-2. To assess antiviral responses, we measured the levels of interferon (IFN) expression, IFN-stimulated gene expression, and infection with a pseudotyped virus bearing either severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike protein or vesicular stomatitis virus (VSV)-G protein after treatment of cells with both bLF and polyinosinic-polycytidylic acid, an analog of double-stranded RNA that mimics viral infection. Combination treatment of cells with both bLF and polyinosinic-polycytidylic acid increased mRNA and protein expression of several IFN genes (IFNB, IFNL1, and IFNL2) and IFN-stimulated genes (ISG15, MX1, IFITM1, and IFITM3) in Caco-2 cells. However, treatment with bLF alone did not induce an antiviral response. Furthermore, combination treatment suppressed infection of the SARS-CoV-2 pseudotyped virus more efficiently than did bLF treatment alone, even though combination treatment increased the expression of mRNA encoding ACE2. These results indicate that bLF increases the antiviral response associated with the double-stranded RNA-stimulated signaling pathway. Our results also suggest that bLF and double-stranded RNA analogs can be used to treat viral infections, including those caused by SARS-CoV-2.

Discovery Proteomics Analysis Determines That Driver Oncogenes Suppress Antiviral Defense Pathways Through Reduction in Interferon-ß Autocrine Stimulation.

Since the discovery of oncogenes, there has been tremendous interest to understand their mechanistic basis and to develop broadly actionable therapeutics. Some of the most frequently activated oncogenes driving diverse cancers are c-MYC, EGFR, HER2, AKT, KRAS, BRAF, and MEK. Using a reductionist approach, we explored how cellular proteomes are remodeled in isogenic cell lines engineered with or without these driver oncogenes. The most striking discovery for all oncogenic models was the systematic downregulation of scores of antiviral proteins regulated by type 1 interferon. These findings extended to cancer cell lines and patient-derived xenograft models of highly refractory pancreatic cancer and osteosarcoma driven by KRAS and MYC oncogenes. The oncogenes reduced basal expression of and autocrine stimulation by type 1 interferon causing remarkable convergence on common phenotypic and functional profiles. In particular, there was dramatically lower expression of dsRNA sensors including DDX58 (RIG-I) and OAS proteins, which resulted in attenuated functional responses when the oncogenic cells were treated with the dsRNA mimetic, polyI:C, and increased susceptibility to infection with an RNA virus shown using SARS-CoV-2. Our reductionist approach provides molecular and functional insights connected to immune evasion hallmarks in cancers and suggests therapeutic opportunities.

UNRAVELING the ANTIVIRAL ACTIVITY of PLITIDEPSIN by ULTRASTRUCTURAL ANALYSIS

Background: The use of compounds against highly conserved cellular host factors required to complete the replication cycle of distinct viruses such as SARS-CoV-2 offers a common solution to diverse viral threats. This approach is especially relevant for pan-antiviral effects given that viruses converge at intracellular steps such as viral genome replication and protein production. Currently, there are only a limited number of approved drugs involved in targeting intracellular host factors. One of these compounds is plitidiepsin, which has shown a potent preclinical efficacy against SARS-CoV-2 by targeting the host protein eEF1A. Plitidepsin inhibits nucleocapsid viral protein expression and viral induced cytopathic effect in vitro. In addition, it also reduces genomic and subgenomic RNA expression. However, how plitidepsin exerts its antiviral activity remains unknown. Methods: Current models of SARS-CoV-2 replication propose that upon viral fusion, non-structural viral proteins form a replication-transcription complex that associates to compartments with a double membrane vesicle (DMV) morphology that shelters the viral genome replication. Here we have used an electron microcopy analysis to explore the antiviral effect of plitidepsin and its impact on SARS-CoV-2 replication and DMV formation on target Vero E6 cells. Results: This ultrastructural analysis allowed to recapitulate the SARS-CoV-2 infectious life cycle, where evident viral DMV formation was observed as well as viral budding events along with cell-associated viruses. However, in cells treated with plitidepsin at different non-toxic concentrations (0.2 and 0.05 μ M) there was a lack of viral DMV formation and a complete absence of viral particles. Complementary SARS-CoV-2 nucleocapsid and dsRNA immunogold labelling unambiguously confirmed the lack of viral replication in plitidepsin-treated cells. Overall, these data indicate that plitidepsin treatment abrogated the formation of DMVs, and the detection of nucleocapsid or dsRNA viral products. Conclusion: Electron microscopy ultrastructural analysis coupled to immunogold labelling of SARS-CoV-2 products offer a unique approach to understand how antivirals work. This knowledge is key to identify the mechanism of action of promising compounds interfering with host factors whose implication in strategic biological processes can be applied as pan-antiviral strategies.

NEUROPILIN-1 MEDIATES SARS-CoV-2 INFECTION of ASTROCYTES PROMOTING NEURON DYSFUNCTION

Background: SARS-CoV-2 primarily infects the lung but may also damage other organs including the brain, heart, kidney, and intestine. Central nervous system (CNS) disorders include loss of smell and taste, headache, delirium, acute psychosis, seizures, and stroke. Pathological loss of gray matter occurs in SARS-CoV-2 infection but it is unclear whether this is due to direct viral infection, indirect effects associated with systemic inflammation, or both. Methods: We used iPSC-derived brain organoids and primary human astrocytes from cerebral cortex to study direct SARS-CoV-2 infection, as confirmed by Spike and Nucleocapsid immunostaining and RT-qPCR. siRNAs, blocking antibodies, and small molecule inhibitors were used to assess SARS-CoV-2 receptor candidates. Bulk RNA-seq, DNA methylation seq, and Nanostring GeoMx digital spatial profiling were utilized to identify virus-induced changes in host gene expression. Results: Astrocytes were robustly infected by SARS-CoV-2 in brain organoids while neurons and neuroprogenitor cells supported only low-level infection. Based on siRNA knockdowns, Neuropilin-1, not ACE2, functioned as the primary receptor for SARS-CoV-2 in astrocytes. The endolysosomal two-pore channel protein, TPC, also facilitated infection likely through its regulatory effects on endocytosis. Other alternative receptors, including the AXL tyrosine kinase, CD147, and dipeptidyl protease 4 (DPP4), did not function as SARS-CoV-2 receptors in astrocytes. SARS-CoV-2 infection dynamically induced type I, II, and III interferons, and genes involved in Toll-like receptor signaling, MDA5 and RIG-I sensing of double-stranded RNA, and production of inflammatory cytokines. Genes activating apoptosis were also increased. Down-regulated genes included those involved in water, ion and lipid transport, synaptic transmission, and formation of cell junctions. Epigenetic analyses revealed transcriptional changes related to DNA methylation states, particularly decreased DNA methylation in interferon-related genes. Long-term viral infection of brain organoids resulted in progressive neuronal degeneration and death. Conclusion: Our findings support a model where SARS-CoV-2 infection of astrocytes produces a panoply of changes in the expression of genes regulating innate immune signaling and inflammatory responses. Deregulation of these genes in astrocytes produces a microenvironment within the CNS that ultimately disrupts normal neuron function, promoting neuronal cell death and CNS deficits.

The Role of Nanotechnology in Antiviral Regime: An Overview

Nanomedicine or nanotechnology exhibits outstanding features to challenge severe health issues including pathogenic viral infections, the most culpable invaders in the present situation. The perpetual mutational pattern in viruses topped with raising resistance to drug epitomizes the current situation as a trigger to explore nanotech platforms in antiviral therapies. Referring to novel physicochemical features of nanomaterials associated with effective drug delivery, it is viewed as an ideal strategy for treatment of viral infections. The coronavirus induced pathogenesis, including MERS, SARS and SARS-CoV-2 infections, has triggered alarming and highly dangerous precedents against existence of humans. Applications of nanotechnology can serve a new direction for disinfection or treatment of viruses. Presently, various types of nanomaterials, such as nanogels, nanospheres, nanocapsules, liposomes, nanoparticles and many others, that have been investigated in vivo and in vitro for successful drug delivery, vaccination, diagnostic assay and device development with anticipation to be translated in advanced clinical practices, need a collective relook. This paper intents to contribute insightful critique of current studies on the efficacy of nanoplatforms as drug transporter, diagnostic tool and vaccine candidate against pathogenic viruses counting the highly pathogenic and incurable "coronaviruses".

Features of innate and adaptive immunity for infections with RNA-containing respiratory viruses

During respiratory infections caused by RNA-containing viruses, double-stranded (ds) replicative forms bind with specific receptors in the endosomes and cytosol of infected cells. Therefore, the induction of gene expression of early cytokines occurs faster and more efficiently dsRNA. Induction of innate and specific adaptive immunity usually provides complete eradication of extracellular virions and infected cells without chronic infection and risk of recombination between viral RNA and host cell chromosomes due to absence of DNA copies of the virus genomes. However, this increases the risk of a cytokine storm with pathological disorders of defense systems. Mutual adaptation of viruses and their hosts includes interaction of viral antigens with host cytokines or corresponding receptors that results in disturbances of the fist line of host defense and infection progression.

Reduced A-to-I editing of endogenous Alu RNAs in lung after SARS-CoV-2 infection.

Due to potential severity of disease caused by SARS-CoV-2 infection, it is critical to understand both mechanisms of viral pathogenesis as well as diversity of host responses to infection. Reduced A-to-I editing of endogenous double-stranded RNAs (dsRNAs), as a result of inactivating mutations in ADAR, produces one form of Aicardi-Goutières Syndrome, with an immune response similar to an anti-viral response. By analyzing whole genome RNA sequencing data, we find reduced levels of A-to-I editing of endogenous Alu RNAs in normal human lung cells after infection by SARS-CoV-2 as well as in lung biopsies from patients with SARS-CoV-2 infections. Unedited Alu RNAs, as seen after infection, induce IRF and NF-kB transcriptional responses and downstream target genes, while edited Alu RNAs as seen in the absence of infection, fail to activate these transcriptional responses. Thus, decreased A-to-I editing may represent an important host response to SARS-CoV-2 infection.