Deleterious single nucleotide polymorphisms (SNPs) of human IFNAR2 gene facilitate COVID-19 severity in patients: a comprehensive in silico approach.

In humans, the dimeric receptor complex IFNAR2-IFNAR1 accelerates cellular response triggered by type I interferon (IFN) family proteins in response to viral infection including Coronavirus infection. Studies have revealed the association of the IFNAR2 gene with severe illness in Coronavirus infection and indicated the association of genomic variants, i.e. single nucleotide polymorphisms (SNPs). However, comprehensive analysis of SNPs of the IFNAR2 gene has not been performed in both coding and non-coding region to find the causes of loss of function of IFNAR2 in COVID-19 patients. In this study, we have characterized coding SNPs (nsSNPs) of IFNAR2 gene using different bioinformatics tools and identified deleterious SNPs. We found 9 nsSNPs as pathogenic and disease-causing along with a decrease in protein stability. We employed molecular docking analysis that showed 5 nsSNPs to decrease binding affinity to IFN. Later, MD simulations showed that P136R mutant may destabilize crucial binding with the IFN molecule in response to COVID-19. Thus, P136R is likely to have a high impact on disrupting the structure of the IFNAR2 protein. GTEx portal analysis predicted 14 sQTLs and 5 eQTLs SNPs in lung tissues hampering the post-transcriptional modification (splicing) and altering the expression of the IFNAR2 gene. sQTLs and eQTLs SNPs potentially explain the reduced IFNAR2 production leading to severe diseases. These mutants in the coding and non-coding region of the IFNAR2 gene can help to recognize severe illness due to COVID 19 and consequently assist to develop an effective drug against the infection.Communicated by Ramaswamy H. Sarma.

A natural goldmine of binding proteins and soluble receptors simplified their translation to blockbuster drugs, all in one decade.

Human urinary proteins are a goldmine of natural proteins a feature that simplifies their translation to biologics. Combining this goldmine together with the ligand-affinity-chromatography (LAC) purification method, proved a winning formula in their isolation. LAC specificity, efficiency, simplicity and inherent indispensability in the search for predictable and unpredictable proteins, is superior to other separation techniques. Unlimited amounts of recombinant cytokines and monoclonal antibodies (mAb) accelerated the "triumph". My approach concluded 35 years of worldwide pursuit for Type I IFN receptor (IFNAR2) and advanced the understanding of the signal transduction of this Type of IFN. TNF, IFNγ and IL-6 as baits enabled the isolation of their corresponding soluble receptors and N-terminal amino acid sequence of the isolated proteins facilitated the cloning of their cell surface counterparts. IL-18, IL-32, and heparanase as the baits yielded the corresponding unpredictable proteins: the antidote IL-18 Binding Protein (IL-18BP), the enzyme Proteinase 3 (PR3) and the hormone Resistin. IFNß proved beneficial in Multiple Sclerosis and is a blockbuster drug, Rebif®. TNF mAbs translated into Remicade® to treat Crohn's disease. Enbrel® based on TBPII is for Rheumatoid Arthritis. Both are blockbusters. Tadekinig alfa™, a recombinant IL-18BP, is in phase III clinical study for inflammatory and autoimmune diseases. Seven years of continuous compassionate use of Tadekinig alfa™ in children born with mutations (NLRC4, XIAP) proved life-saving and is an example of tailored made medicine. IL-18 is a checkpoint biomarker in cancer and IL-18BP is planned recently to target cytokine storms resulting from CAR-T treatment and in COVID 19.

Polymorphisms in ACE1, TMPRSS2, IFIH1, IFNAR2, and TYK2 Genes Are Associated with Worse Clinical Outcomes in COVID-19.

Although advanced age, male sex, and some comorbidities impact the clinical course of COVID-19, these factors only partially explain the inter-individual variability in disease severity. Some studies have shown that genetic polymorphisms contribute to COVID-19 severity; however, the results are inconclusive. Thus, we investigated the association between polymorphisms in ACE1, ACE2, DPP9, IFIH1, IFNAR2, IFNL4, TLR3, TMPRSS2, and TYK2 and the clinical course of COVID-19. A total of 694 patients with COVID-19 were categorized as: (1) ward inpatients (moderate symptoms) or patients admitted at the intensive care unit (ICU; severe symptoms); and (2) survivors or non-survivors. In females, the rs1990760/IFIH1 T/T genotype was associated with risk of ICU admission and death. Moreover, the rs1799752/ACE1 Ins and rs12329760/TMPRSS2 T alleles were associated with risk of ICU admission. In non-white patients, the rs2236757/IFNAR2 A/A genotype was associated with risk of ICU admission, while the rs1799752/ACE1 Ins/Ins genotype, rs2236757/IFNAR2 A/A genotype, and rs12329760/TMPRSS2 T allele were associated with risk of death. Moreover, some of the analyzed polymorphisms interact in the risk of worse COVID-19 outcomes. In conclusion, this study shows an association of rs1799752/ACE1, rs1990760/IFIH1, rs2236757/IFNAR2, rs12329760/TMPRSS2, and rs2304256/TYK2 polymorphisms with worse COVID-19 outcomes, especially among female and non-white patients.

Construction and Transcriptomic Study of Chicken IFNAR1-Knockout Cell Line Reveals the Essential Roles of Cell Growth- and Apoptosis-Related Pathways in Duck Tembusu Virus Infection.

For industrial vaccine production, overwhelming the existing antiviral innate immune response dominated by type I interferons (IFN-I) in cells would be a key factor improving the effectiveness and production cost of vaccines. In this study, we report the construction of an IFN-I receptor 1 (IFNAR1)-knockout DF-1 cell line (KO-IFNAR1), which supports much more efficient replication of the duck Tembusu virus (DTMUV), Newcastle disease virus (NDV) and gammacoronavirus infectious bronchitis virus (IBV). Transcriptomic analysis of DTMUV-infected KO-IFNAR1 cells demonstrated that DTMUV mainly activated genes and signaling pathways related to cell growth and apoptosis. Among them, JUN, MYC and NFKBIA were significantly up-regulated. Furthermore, knockdown of zinc-fingered helicase 2 (HELZ2) and interferon-α-inducible protein 6 (IFI6), the two genes up-regulated in both wild type and KO-IFNAR1 cells, significantly increased the replication of DTMUV RNA. This study paves the way for further studying the mechanism underlying the DTMUV-mediated IFN-I-independent regulation of virus replication, and meanwhile provides a potential cell resource for efficient production of cell-based avian virus vaccines.

IFNAR2 relevance in the clinical outcome of individuals with severe COVID-19.

Interferons (IFNs) are a group of cytokines with antiviral, antiproliferative, antiangiogenic, and immunomodulatory activities. Type I IFNs amplify and propagate the antiviral response by interacting with their receptors, IFNAR1 and IFNAR2. In COVID-19, the IFNAR2 (interferon alpha and beta receptor subunit 2) gene has been associated with the severity of the disease, but the soluble receptor (sIFNAR2) levels have not been investigated. We aimed to evaluate the association of IFNAR2 variants (rs2236757, rs1051393, rs3153, rs2834158, and rs2229207) with COVID-19 mortality and to assess if there was a relation between the genetic variants and/or the clinical outcome, with the levels of sIFNAR2 in plasma samples from hospitalized individuals with severe COVID-19. We included 1,202 subjects with severe COVID-19. The genetic variants were determined by employing Taqman® assays. The levels of sIFNAR2 were determined with ELISA in plasma samples from a subgroup of 351 individuals. The rs2236757, rs3153, rs1051393, and rs2834158 variants were associated with mortality risk among patients with severe COVID-19. Higher levels of sIFNAR2 were observed in survivors of COVID-19 compared to the group of non-survivors, which was not related to the studied IFNAR2 genetic variants. IFNAR2, both gene, and soluble protein, are relevant in the clinical outcome of patients hospitalized with severe COVID-19.

Herbal medications and natural products for patients with covid-19 and diabetes mellitus: Potentials and challenges.

Background: The presence of diabetes mellitus (DM) among COVID-19 patients is associated with increased hospitalization, morbidity, and mortality. Evidence has shown that hyperglycemia potentiates SARS-CoV-2 (severe acute respiratory syndrome coronavirus 2) infection and plays a central role in severe COVID-19 and diabetes comorbidity. In this review, we explore the therapeutic potentials of herbal medications and natural products in the management of COVID-19 and DM comorbidity and the challenges associated with the preexisting or concurrent use of these substances. Methods: Research papers that were published from January 2016 to December 2021 were retrieved from PubMed, ScienceDirect, and Google Scholar databases. Papers reporting clinical evidence of antidiabetic activities and any available evidence of the anti-COVID-19 potential of ten selected natural products were retrieved and analyzed for discussion in this review. Results: A total of 548 papers (73 clinical trials on the antidiabetic activities of the selected natural products and 475 research and review articles on their anti-COVID-19 potential) were retrieved from the literature search for further analysis. A total of 517 articles (reviews and less relevant research papers) were excluded. A cumulative sum of thirty-one (31) research papers (20 clinical trials and 10 others) met the criteria and have been discussed in this review. Conclusion: The findings of this review suggest that phenolic compounds are the most promising phytochemicals in the management of COVID-19 and DM comorbidity. Curcumin and propolis have shown substantial evidence against COVID-19 and DM in humans and are thus, considered the best potential therapeutic options.

SARS-Cov2 acute and post-active infection in the context of autoimmune and chronic inflammatory diseases.

The clinical and immunological spectrum of acute and post-active COVID-19 syndrome overlaps with criteria used to characterize autoimmune diseases such as rheumatoid arthritis (RA) and systemic lupus erythematosus (SLE). Indeed, following SARS-Cov2 infection, the innate immune response is altered with an initial delayed production of interferon type I (IFN-I), while the NF-kappa B and inflammasome pathways are activated. In lung and digestive tissues, an alternative and extrafollicular immune response against SARS-Cov2 takes place with, consequently, an altered humoral and memory T cell response leading to breakdown of tolerance with the emergence of autoantibodies. However, the risk of developing severe COVID-19 among SLE and RA patients did not exceed the general population except in those having pre-existing neutralizing autoantibodies against IFN-I. Treatment discontinuation rather than COVID-19 infection or vaccination increases the risk of developing flares. Last but not least, a limited number of case reports of individuals having developed SLE or RA following COVID-19 infection/vaccination have been reported. Altogether, the SARS-Cov2 pandemic represents an unique opportunity to investigate the dangerous interplay between the immune response against infectious agents and autoimmunity, and to better understand the triggering role of infection as a risk factor in autoimmune and chronic inflammatory disease development.

Expression analysis of IFNAR1 and TYK2 transcripts in COVID-19 patients.

As a member of JAK family of non-receptor tyrosine kinases, TYK2 has a crucial role in regulation of immune responses. This protein has a crucial role in constant expression of IFNAR1 on surface of cells and initiation of type I IFN signaling. In the current study, we measured expression of IFNAR1 and TYK2 levels in venous blood samples of COVID-19 patients and matched controls. TYK2 was significantly down-regulated in male patients compared with male controls (RME = 0.34, P value = 0.03). Though, levels of TYK2 were not different between female cases and female controls, or between ICU-admitted and non-ICU-admitted cases. Expression of IFNAR1 was not different either between COVID-19 cases and controls or between patients required ICU admission and non-ICU-admitted cases. However, none of these transcripts can properly diffrentiate COVID-19 cases from controls or separate patients based on disease severity. The current study proposes down-regulation of TYK2 as a molecular mechanism for incapacity of SARS-CoV-2 in induction of a competent IFN response.

Inherited IFNAR1 Deficiency in a Child with Both Critical COVID-19 Pneumonia and Multisystem Inflammatory Syndrome.

BACKGROUND: Inborn errors of immunity (IEI) and autoantibodies to type I interferons (IFNs) underlie critical COVID-19 pneumonia in at least 15% of the patients, while the causes of multisystem inflammatory syndrome in children (MIS-C) remain elusive. OBJECTIVES: To detect causal genetic variants in very rare cases with concomitant critical COVID-19 pneumonia and MIS-C. METHODS: Whole exome sequencing was performed, and the impact of candidate gene variants was investigated. Plasma levels of cytokines, specific antibodies against the virus, and autoantibodies against type I IFNs were also measured. RESULTS: We report a 3-year-old child who died on day 56 of SARS-CoV-2 infection with an unusual clinical presentation, combining both critical COVID-19 pneumonia and MIS-C. We identified a large, homozygous loss-of-function deletion in IFNAR1, underlying autosomal recessive IFNAR1 deficiency. CONCLUSIONS: Our findings confirm that impaired type I IFN immunity can underlie critical COVID-19 pneumonia, while suggesting that it can also unexpectedly underlie concomitant MIS-C. Our report further raises the possibility that inherited or acquired dysregulation of type I IFN immunity might contribute to MIS-C in other patients.

Interferon Inhibition Enhances the Pilot-Scale Production of Rabies Virus in Human Diploid MRC-5 Cells.

Inactivated vaccines based on cell culture are very useful in the prevention and control of many diseases. The most popular strategy for the production of inactivated vaccines is based on monkey-derived Vero cells, which results in high productivity of the virus but has a certain carcinogenic risk due to non-human DNA contamination. Since human diploid cells, such as MRC-5 cells, can produce a safer vaccine, efforts to develop a strategy for inactivated vaccine production using these cells have been investigated using MRC-5 cells. However, most viruses do not replicate efficiently in MRC-5 cells. In this study, we found that rabies virus (RABV) infection activated a robust interferon (IFN)-ß response in MRC-5 cells but almost none in Vero cells, suggesting that the IFN response could be a key limiting factor for virus production. Treatment of the MRC-5 cells with IFN inhibitors increased RABV titers by 10-fold. Additionally, the RABV titer yield was improved five-fold when using IFN receptor 1 (IFNAR1) antibodies. As such, we established a stable IFNAR1-deficient MRC-5 cell line (MRC-5IFNAR1-), which increased RABV production by 6.5-fold compared to normal MRC-5 cells. Furthermore, in a pilot-scale production in 1500 square centimeter spinner flasks, utilization of the MRC-5IFNAR1- cell line or the addition of IFN inhibitors to MRC cells increased RABV production by 10-fold or four-fold, respectively. Thus, we successfully established a human diploid cell-based pilot scale virus production platform via inhibition of IFN response for rabies vaccines, which could also be used for other inactivated virus vaccine production.

All hands on deck: SARS-CoV-2 proteins that block early anti-viral interferon responses.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection is responsible for the current pandemic coronavirus disease of 2019 (COVID-19). Like other pathogens, SARS-CoV-2 infection can elicit production of the type I and III interferon (IFN) cytokines by the innate immune response. A rapid and robust type I and III IFN response can curb viral replication and improve clinical outcomes of SARS-CoV-2 infection. To effectively replicate in the host, SARS-CoV-2 has evolved mechanisms for evasion of this innate immune response, which could also modulate COVID-19 pathogenesis. In this review, we discuss studies that have reported the identification and characterization of SARS-CoV-2 proteins that inhibit type I IFNs. We focus especially on the mechanisms of nsp1 and ORF6, which are the two most potent and best studied SARS-CoV-2 type I IFN inhibitors. We also discuss naturally occurring mutations in these SARS-CoV-2 IFN antagonists and the impact of these mutations in vitro and on clinical presentation. As SARS-CoV-2 continues to spread and evolve, researchers will have the opportunity to study natural mutations in IFN antagonists and assess their role in disease. Additional studies that look more closely at previously identified antagonists and newly arising mutants may inform future therapeutic interventions for COVID-19.

A Case of Autosomal Recessive Interferon Alpha/Beta Receptor Alpha Chain (IFNAR1) Deficiency with Severe COVID-19.

BACKGROUND: Interferons (IFNs) play a crucial role in antiviral immunity. Genetic defects in interferon receptors, IFNs, and auto-antibodies against IFNs can lead to the development of life-threatening forms of infectious diseases like a severe form of COVID-19. CASE PRESENTATION: A 13-year-old boy with a previously reported homozygous loss-of-function mutation in interferon alpha/beta receptor subunit 1 (IFNAR1) (c.674-2A > G) was diagnosed with severe COVID-19. He had cold symptoms and a high-grade fever at the time of admission. He was admitted to the pediatric intensive care unit after showing no response to favipiravir and being hypoxemic. High-resolution computed tomography (HRCT) scanning revealed lung involvement of 70% with extensive areas of consolidation in both lungs. Antibiotics, interferon gamma (IFN-γ), remdesivir, methylprednisolone pulse, and other medications were started in the patient. However, remdesivir and methylprednisolone pulse were discontinued because of their adverse side effects in the patient. His general condition improved, and a few days later was discharged from the hospital. CONCLUSION: We reported a patient with severe COVID-19 who had a mutation in IFNAR1. Our finding suggests that patients with IFNAR1 deficiency are prone to severe forms of COVID-19. Besides, IFN-γ therapy may be a potential drug to treat patients with defects in IFN-α/ß signaling pathways which needs further investigations.

Hypotheses and facts for genetic factors related to severe COVID-19.

Genome-wide association analysis allows the identification of potential candidate genes involved in the development of severe coronavirus disease 2019 (COVID-19). Hence, it seems that genetics matters here, as well. Nevertheless, the virus's nature, including its RNA structure, determines the rate of mutations leading to new viral strains with all epidemiological and clinical consequences. Given these observations, we herein comment on the current hypotheses about the possible role of the genes in association with COVID-19 severity. We discuss some of the major candidate genes that have been identified as potential genetic factors associated with the COVID-19 severity and infection susceptibility: HLA, ABO, ACE2, TLR7, ApoE, TYK2, OAS, DPP9, IFNAR2, CCR2, etc. Further study of genes and genetic variants will be of great benefit for the prevention and assessment of the individual risk and disease severity in different populations. These scientific data will serve as a basis for the development of clinically applicable diagnostic and prognostic tests for patients at high risk of COVID-19.

Mendelian randomization analysis identified genes pleiotropically associated with the risk and prognosis of COVID-19.

OBJECTIVES: COVID-19 has caused a large global pandemic. Patients with COVID-19 exhibited considerable variation in disease behavior. Pervious genome-wide association studies have identified potential genetic variants involved in the risk and prognosis of COVID-19, but the underlying biological interpretation remains largely unclear. METHODS: We applied the summary data-based Mendelian randomization (SMR) method to identify genes that were pleiotropically associated with the risk and various outcomes of COVID-19, including severe respiratory confirmed COVID-19 and hospitalized COVID-19. RESULTS: In blood, we identified 2 probes, ILMN_1765146 and ILMN_1791057 tagging IFNAR2, that showed pleiotropic association with hospitalized COVID-19 (ß [SE]=0.42 [0.09], P = 4.75 × 10-06 and ß [SE]=-0.48 [0.11], P = 6.76 × 10-06, respectively). Although no other probes were significant after correction for multiple testing in both blood and lung, multiple genes as tagged by the top 5 probes were involved in inflammation or antiviral immunity, and several other tagged genes, such as PON2 and HPS5, were involved in blood coagulation. CONCLUSIONS: We identified IFNAR2 and other potential genes that could be involved in the susceptibility or prognosis of COVID-19. These findings provide important leads to a better understanding of the mechanisms of cytokine storm and venous thromboembolism in COVID-19 and potential therapeutic targets for the effective treatment of COVID-19.

Characterization of local SARS-CoV-2 isolates and pathogenicity in IFNAR-/- mice.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) recently a global pandemic with unprecedented public health, economic and social impact. The development of effective mitigation strategies, therapeutics and vaccines relies on detailed genomic and biological characterization of the regional viruses. This study was carried out to isolate SARS-CoV-2 viruses circulating in Anatolia, and to investigate virus propagation in frequently-used cells and experimental animals. We obtained two SARS-CoV-2 viruses from nasopharngeal swabs of confirmed cases in Vero E6 cells, visualized the virions using atomic force and scanning electron microscopy and determined size distribution of the particles. Viral cytopathic effects on Vero E6 cells were initially observed at 72 h post-inoculation and reached 90% of the cells on the 5th day. The isolates displayed with similar infectivity titers, time course and infectious progeny yields. Genome sequencing revealed the viruses to be well-conserved, with less than 1% diversity compared to the prototype virus. The analysis of the viral genomes, along with the available 62 complete genomes from Anatolia, showed limited diversity (up to 0.2% on deduced amino acids) and no evidence of recombination. The most prominent sequence variation was observed on the spike protein, resulting in the substitution D614G, with a prevalence of 56.2%. The isolates produced non-fatal infection in the transgenic type I interferon knockout (IFNAR-/-) mice, with varying neutralizing antibody titers. Hyperemia, regional consolidation and subpleural air accumulation was observed on necropsy, with similar histopathological and immunohistochemistry findings in the lungs, heart, stomach, intestines, liver, spleen and kidneys. Peak viral loads were detected in the lungs, with virus RNA present in the kidneys, jejunum, liver, spleen and heart. In conclusion, we characterized two local isolates, investigated in vitro growth dynamics in Vero E6 cells and identified IFNAR-/- mice as a potential animal model for SARS-CoV-2 experiments.

Neuronal Ablation of Alpha/Beta Interferon (IFN-α/ß) Signaling Exacerbates Central Nervous System Viral Dissemination and Impairs IFN-γ Responsiveness in Microglia/Macrophages.

Alpha/beta interferon (IFN-α/ß) signaling through the IFN-α/ß receptor (IFNAR) is essential to limit virus dissemination throughout the central nervous system (CNS) following many neurotropic virus infections. However, the distinct expression patterns of factors associated with the IFN-α/ß pathway in different CNS resident cell populations implicate complex cooperative pathways in IFN-α/ß induction and responsiveness. Here we show that mice devoid of IFNAR1 signaling in calcium/calmodulin-dependent protein kinase II alpha (CaMKIIα) expressing neurons (CaMKIIcre:IFNARfl/fl mice) infected with a mildly pathogenic neurotropic coronavirus (mouse hepatitis virus A59 strain [MHV-A59]) developed severe encephalomyelitis with hind-limb paralysis and succumbed within 7 days. Increased virus spread in CaMKIIcre:IFNARfl/fl mice compared to IFNARfl/fl mice affected neurons not only in the forebrain but also in the mid-hind brain and spinal cords but excluded the cerebellum. Infection was also increased in glia. The lack of viral control in CaMKIIcre:IFNARfl/fl relative to control mice coincided with sustained Cxcl1 and Ccl2 mRNAs but a decrease in mRNA levels of IFNα/ß pathway genes as well as Il6, Tnf, and Il1ß between days 4 and 6 postinfection (p.i.). T cell accumulation and IFN-γ production, an essential component of virus control, were not altered. However, IFN-γ responsiveness was impaired in microglia/macrophages irrespective of similar pSTAT1 nuclear translocation as in infected controls. The results reveal how perturbation of IFN-α/ß signaling in neurons can worsen disease course and disrupt complex interactions between the IFN-α/ß and IFN-γ pathways in achieving optimal antiviral responses.IMPORTANCE IFN-α/ß induction limits CNS viral spread by establishing an antiviral state, but also promotes blood brain barrier integrity, adaptive immunity, and activation of microglia/macrophages. However, the extent to which glial or neuronal signaling contributes to these diverse IFN-α/ß functions is poorly understood. Using a neurotropic mouse hepatitis virus encephalomyelitis model, this study demonstrated an essential role of IFN-α/ß receptor 1 (IFNAR1) specifically in neurons to control virus spread, regulate IFN-γ signaling, and prevent acute mortality. The results support the notion that effective neuronal IFNAR1 signaling compensates for their low basal expression of genes in the IFN-α/ß pathway compared to glia. The data further highlight the importance of tightly regulated communication between the IFN-α/ß and IFN-γ signaling pathways to optimize antiviral IFN-γ activity.