Highly Sensitive Electrochemiluminescence Biosensing Method for SARS-CoV-2 N Protein Incorporating the Micelle Probes of Quantum Dots and Dibenzoyl Peroxide Using the Screen-Printed Carbon Electrode Modified with a Carboxyl-Functionalized Graphene.

Obtaining stable electrochemiluminescence (ECL) emissions from a hydrophobic luminophore in aqueous solutions and designing a method without the use of an exogenous coreactant are promising for ECL biosensing. Here, a highly sensitive signal-on ECL immunoassay for the SARS-CoV-2 N protein was developed using micelles as an ECL tag. The micelles were prepared by coencapsulating the luminophore hydrophobic CdSe/ZnS quantum dots and coreactant dibenzoyl peroxide within the hydrophobic core of micelles. The ECL probe was obtained by covalently bonding a SARS-CoV-2 N protein-binding aptamer onto the micelle surface. The construction of the immunosensor was initiated by the immobilization of the anti-SARS-CoV-2 N protein antibody onto the screen-printed carbon electrode (SPCE) with a -COOH-functionalized surface. The surface functionalization of SPCEs was achieved through paste-exfoliated graphene, which was modified with a -COOH group through supramolecular-covalent scaffolds on SPCE. Upon achieving sandwich complexes on the immunosensor, an efficient ECL signal response at -1.4 V versus Ag/AgCl was obtained in phosphate buffer solution. The ECL assay was used for the sensitive determination of SARS-CoV-2 N protein with the linear range from 0.01 to 50 ng mL-1, and the detection limit was 3.0 pg mL-1. The immunosensor showed good reproducibility and stability, and the ECL immunoassay was used to determine the SARS-CoV-2 N protein in serum samples. The proposed approach to obtain micelles is versatile for the preparation of stable ECL luminophores by using hydrophobic materials, and the strategy provides an alternative for ECL bioassays based on the coreactant route.

Modified exfoliated graphene functionalized with carboxylic acid-group and thionine on a screen-printed carbon electrode as a platform for an electrochemical enzyme immunosensor.

An enzyme immunoassay was developed based on the coulometric measurement of immunoglobulin M (IgM) against Hantaan viruses (HTNV) by using virus-like particles (VLPs) as recognition molecules. The surface functionalization of screen-printed carbon electrodes (SPCEs) was achieved through paste-exfoliated graphene that was modified with a COOH group and a thionine mediator through supramolecular-covalent scaffolds, on SPCEs by using the binder contained in the ink. After the covalent immobilization of the antibody, the sensor was used for the sandwich enzyme immunoassay of IgM against HTNV. By using HTNV VLPs as the second recognization molecules, the resulting sensor efficiently monitored the reaction of IgM against HTNV and anti-IgM antibody with high specificity. By attaching HTNV nucleocapsid protein antibody conjugate with horseradish peroxidase (HRP) onto VLPs, the signal response of the assay was derived from the coulometric measurement of H2O2 reduction mediated by thionine on the electrode surface after the application of a potential (- 0.2 V vs. Ag/AgCl). The ratio of charges measured before or after H2O2 addition was used to quantify IgM because these charges could be used as background charges or total charges, respectively. The ratio exhibited good agreement with IgM concentration within a range 0.1 to 1000 pg mL-1, and a detection limit of 0.06 pg mL-1 was obtained. The assay demonstrated high sensitivity and specificity toward HTNV-specific IgM in serum.

N6-methyladenosine modifications enhance enterovirus 71 ORF translation through METTL3 cytoplasmic distribution.

During replication, numerous viral RNAs are modified by N6-methyladenosine (m6A), the most abundant internal RNA modification. m6A is believed to regulate elements of RNA metabolism, such as splicing, stability, translation, secondary structure formation, and viral replication. In this study, we assessed the occurrence of m6A modification of the EV71 genome in human cells and revealed a preferred, conserved modification site across diverse viral strains. A single m6A modification at the 5' UTR-VP4 junction was shown to perform a protranslational function. Depletion of the METTL3 methyltransferase or treatment with 3-deazaadenosine significantly reduced EV71 replication. Specifically, METTL3 colocalized with the viral dsRNA replication intermediate in the cytoplasm during EV71 infection. As a nuclear resident protein, METTL3 relies on the binding of the nuclear import protein karyopherin to its nuclear localization signal (NLS) for nuclear translocation. We observed that EV71 2A and METTL3 share nuclear import proteins. The results of this study revealed an inner mechanism by which EV71 2A regulates the subcellular location of METTL3 to amplify its own gene expression, providing an increased understanding of RNA epitranscriptomics during the EV71 replication cycle.

Protective CD8+ T-cell response against Hantaan virus infection induced by immunization with designed linear multi-epitope peptides in HLA-A2.1/Kb transgenic mice.

BACKGROUND: An effective vaccine that prevents disease caused by hantaviruses is a global public health priority, but up to now, no vaccine has been approved for worldwide use. Therefore, novel vaccines with high prophylaxis efficacy are urgently needed. METHODS: Herein, we designed and synthesized Hantaan virus (HTNV) linear multi-epitope peptide consisting of HLA-A*02-restricted HTNV cytotoxic T cell (CTL) epitope and pan HLA-DR-binding epitope (PADRE), and evaluated the immunogenicity, as well as effectiveness, of multi-epitope peptides in HLA-A2.1/Kb transgenic mice with interferon (IFN)-γ enzyme-linked immunospot assay, cytotoxic mediator detection, proliferation assay and HTNV-challenge test. RESULTS: The results showed that a much higher frequency of specific IFN-γ-secreting CTLs, high levels of granzyme B production, and a strong proliferation capacity of specific CTLs were observed in splenocytes of mice immunized with multi-epitope peptide than in those of a single CTL epitope. Moreover, pre-immunization of multi-epitope peptide could reduce the levels of HTNV RNA loads in the liver, spleen and kidneys of mice, indicating that specific CTL responses induced by multi-epitope peptide could reduce HTNV RNA loads in vivo. CONCLUSIONS: This study may provide an important foundation for the development of novel peptide vaccines for HTNV prophylaxis.

The Long Noncoding RNA NEAT1 Exerts Antihantaviral Effects by Acting as Positive Feedback for RIG-I Signaling.

Hantavirus infection, which causes zoonotic diseases with a high mortality rate in humans, has long been a global public health concern. Over the past decades, accumulating evidence suggests that long noncoding RNAs (lncRNAs) play key regulatory roles in innate immunity. However, the involvement of host lncRNAs in hantaviral control remains uncharacterized. In this study, we identified the lncRNA NEAT1 as a vital antiviral modulator. NEAT1 was dramatically upregulated after Hantaan virus (HTNV) infection, whereas its downregulation in vitro or in vivo delayed host innate immune responses and aggravated HTNV replication. Ectopic expression of NEAT1 enhanced beta interferon (IFN-ß) production and suppressed HTNV infection. Further investigation suggested that NEAT1 served as positive feedback for RIG-I signaling. HTNV infection activated NEAT1 transcription through the RIG-I-IRF7 pathway, whereas NEAT1 removed the transcriptional inhibitory effects of the splicing factor proline- and glutamine-rich protein (SFPQ) by relocating SFPQ to paraspeckles, thus promoting the expression of RIG-I and DDX60. RIG-I and DDX60 had synergic effects on IFN production. Taken together, our findings demonstrate that NEAT1 modulates the innate immune response against HTNV infection, providing another layer of information about the role of lncRNAs in controlling viral infections.IMPORTANCE Hantaviruses have attracted worldwide attention as archetypal emerging pathogens. Recently, increasing evidence has highlighted long noncoding RNAs (lncRNAs) as key regulators of innate immunity; however, their roles in hantavirus infection remain unknown. In the present work, a new unexplored function of lncRNA NEAT1 in controlling HTNV replication was found. NEAT1 promoted interferon (IFN) responses by acting as positive feedback for RIG-I signaling. This lncRNA was induced by HTNV through the RIG-I-IRF7 pathway in a time- and dose-dependent manner and promoted HTNV-induced IFN production by facilitating RIG-I and DDX60 expression. Intriguingly, NEAT1 relocated SFPQ and formed paraspeckles after HTNV infection, which might reverse inhibitive effects of SFPQ on the transcription of RIG-I and DDX60. To the best of our knowledge, this is the first study to address the regulatory role of the lncRNA NEAT1 in host innate immunity after HTNV infection. In summary, our findings provide additional insights regarding the role of lncRNAs in controlling viral infections.

DDX50 inhibits the replication of dengue virus 2 by upregulating IFN-ß production.

Dengue virus (DENV) infects approximately 390 million people per year, and each of the four DENV serotypes (DENV-1, DENV-2, DENV-3, and DENV-4) is capable of causing infection. At present, there is no antiviral drug available for the treatment of DENV. Several DExD/H-box helicases have been shown to be involved in the antiviral immune response or viral replication. In the present study, we investigated the role of DDX50 in DENV-2 RNA replication. Our data showed that the level of DENV-2 RNA increased in DDX50 knockdown cells during an early stage of viral infection and decreased in DDX50-overexpressing cells. DDX50, in conjunction with RIG-I and MDA5, upregulated the production of IFN-ß in infected cells through an additive effect on the IFN-ß promoter. Furthermore, transcription of several IFN-stimulated genes was increased in DDX50-overexpressing cells infected with DENV-2. These results provide evidence that DDX50 negatively regulates DENV-2 replication during the early stages of infection by inducing IFN-ß production.

Thermodynamic and kinetic investigation on the crucial factors affecting adefovir dipivoxil-saccharin cocrystallization.

PURPOSE: The aim of this study was to perform a thermodynamic and kinetic investigation on the crucial factors affecting the cocrystallization between adefovir dipivoxil (AD) and saccharin (SAC). METHODS: Phase solubility diagrams and ternary phase diagrams were constructed based on the solubility data of AD, SAC and their cocrystals in ethanol, isopropanol and ethyl acetate at different temperatures. The conductimetric method was used to determine the induction time. A quantitative and intuitive technique modified from dissolution testing was employed to investigate the cocrystallization kinetics. RESULTS: AD-SAC cocrystals exhibited different crystal habits but only one cocrystal polymorph was confirmed. The effects of several crucial factors, including the input amounts of two components, AD/SAC ratio, solvent and temperature, on the crystallization of single-component alone, cocrystal formation, cocrystal stability, supersaturation, nucleation, crystal growth and cocrystal yield were determined. Thermodynamic and kinetic parameters provided the rationale for this spontaneous cocrystallization system without the need of solvent evaporation and temperature change. CONCLUSIONS: This systemic investigation enriched the present understanding of thermodynamics and kinetics of cocrystals and built the groundwork for AD-SAC cocrystal scale-up.

[Exploring a novel instructional paradigm for the Fundamentals of Immunity and Infection module in our university: A study based on the O-PIRTAS flipped classroom approach].

Medical Microbiology and Medical Immunology are important components of our university's the modular teaching on fundamentals of immunity and infection. Among these, Bacterial Infection and Immunity serves as a bridge between Medical Microbiology and Medical Immunology. This chapter mainly introduces how pathogenic bacteria invade the body to cause infection and how the body's immune system resists bacterial infection. Studying this chapter, students can build a framework knowledge on infection-immunity. However, due to the complexity of the content and the limited duration of the course, the traditional teaching method struggles to help students clarify the knowledge structure, resulting in poor learning outcomes. Therefore, there is an urgent need for reforms. Using the bacterial infection and immunity chapter as an example, this article explores the teaching reform of the Fundamentals of Immunology and Infection module based on the O-PIRTAS flipped classroom model, providing valuable insights for subsequent teaching reforms.

Genomic sequencing revealed recombination event between clade 1 and clade 2 occurs in circulating varicella-zoster virus in China.

Varicella-zoster virus (VZV), a member of the Alphaherpesvirinae subfamily, causes varicella in primary infections and establishing a latent stage in sensory ganglia. Upon reactivation, VZV causes herpes zoster with severe neuralgia, especially in elderly patients. The mutation rate for VZV is comparatively lower than the other members of other alpha herpesviruses. Due to geographic isolation, different genotypes of VZV are circulating on separate continents. Here, we successfully isolated a VZV from the vesicular fluid of a youth zoster patient. Based on the single-nucleotide polymorphism profiles of different open reading frames that define the genotype, this newly isolated VZV primarily represents genotype clade 2 but also has characteristics of genotype clade 1. The next-generation sequencing provided a nearly full-length sequence, and further phylogenetic analysis revealed that this VZV isolate is distinct from clades 1 and 2. The Recombination Detection Program indicates that a possible recombinant event may occur between the VZV isolate and clade 1. In summary, we found that there is a circulating VZV isolate in China that may represent a recombinant between clade 1 and clade 2, providing new concerns that need to be considered in the future VZV vaccination program.

Disparate macrophage responses are linked to infection outcome of Hantan virus in humans or rodents.

Hantaan virus (HTNV) is asymptomatically carried by rodents, yet causes lethal hemorrhagic fever with renal syndrome in humans, the underlying mechanisms of which remain to be elucidated. Here, we show that differential macrophage responses may determine disparate infection outcomes. In mice, late-phase inactivation of inflammatory macrophage prevents cytokine storm syndrome that usually occurs in HTNV-infected patients. This is attained by elaborate crosstalk between Notch and NF-κB pathways. Mechanistically, Notch receptors activated by HTNV enhance NF-κB signaling by recruiting IKKß and p65, promoting inflammatory macrophage polarization in both species. However, in mice rather than humans, Notch-mediated inflammation is timely restrained by a series of murine-specific long noncoding RNAs transcribed by the Notch pathway in a negative feedback manner. Among them, the lnc-ip65 detaches p65 from the Notch receptor and inhibits p65 phosphorylation, rewiring macrophages from the pro-inflammation to the pro-resolution phenotype. Genetic ablation of lnc-ip65 leads to destructive HTNV infection in mice. Thus, our findings reveal an immune-braking function of murine noncoding RNAs, offering a special therapeutic strategy for HTNV infection.

A comprehensive investigation of Glycoprotein-based nucleic acid vaccines for Hantaan Virus.

Hemorrhagic fever with renal syndrome (HFRS) occurs throughout Eurasia with considerable morbidity and mortality. Currently, the absence of specific treatments or effective antiviral drugs for hantavirus infection makes developing safe and effective vaccines a high priority. Here, we report the development of three novel nucleic acid vaccine candidates, mRNA, naked DNA, and DNA encapsulated in lipid nanoparticles, encoding the glycoproteins of the Hantaan virus (HTNV). To comprehensively evaluate the potential of candidate HTNV nucleic acid vaccines in preventing HFRS, we focus on evaluating their immunogenicity and efficacy in mice and comparing them with an inactivated vaccine as the benchmark. Our findings reveal that all candidate vaccines activated instant and sustained immune responses, offering comparable in vivo protective efficacy to the inactivated vaccines. Notably, compared to the inactivated vaccine, mRNA vaccine induced stronger virus-specific T-helper 1 cell immune response, while DNA-LNP elicited higher levels of neutralizing antibodies in mice. These results mark a significant step in developing nucleic acid vaccines for HTNV, suggesting that sequential immunization with DNA and mRNA vaccines could further amplify the advantages of nucleic acid vaccines.

Single dose recombinant VSV based vaccine elicits robust and durable neutralizing antibody against Hantaan virus.

Hantaan virus (HTNV) is a pathogenic orthohantavirus prevalent in East Asia that is known to cause hemorrhagic fever with severe renal syndrome (HFRS), which has a high fatality rate. However, a Food and Drug Administration (FDA)-approved vaccine is not currently available against this virus. Although inactivated vaccines have been certified and used in endemic regions for decades, the neutralizing antibody (NAb) titer induced by inactivated vaccines is low and the immunization schedule is complicated, requiring at least three injections spanning approximately 6 months to 1 year. Replication-competent vesicular stomatitis virus (VSV)-based vaccines provide prolonged protection after a single injection. In this study, we successfully engineered the HTNV glycoprotein (GP) in the VSV genome by replacing the VSV-G open reading frame. The resulting recombinant (r) rVSV-HTNV-GP was rescued, and the immunogenicity of GP was similar to that of HTNV. BALB/c mice immunized with rVSV-HTNV-GP showed a high titer of NAb against HTNV after a single injection. Notably, the cross-reactive NAb response induced by rVSV-HTNV-GP against Seoul virus (an orthohantavirus) was higher than that induced by three sequential injections of inactivated vaccines. Upon challenge with HTNV, rVSV-HTNV-GP-immunized mice showed a profoundly reduced viral burden in multiple tissues, and inflammation in the lungs and liver was nearly undetectable. Moreover, a single injection of rVSV-HTNV-GP established a prolonged immunological memory status as the NAbs were sustained for over 1 year and provided long-term protection against HTNV infection. The findings of our study can support further development of an rVSV-HTNV-GP-based HTNV vaccine with a simplified immunization schedule.

DNA Vaccines Encoding HTNV GP-Derived Th Epitopes Benefited from a LAMP-Targeting Strategy and Established Cellular Immunoprotection.

Vaccines has long been the focus of antiviral immunotherapy research. Viral epitopes are thought to be useful biomarkers for immunotherapy (both antibody-based and cellular). In this study, we designed a novel vaccine molecule, the Hantaan virus (HTNV) glycoprotein (GP) tandem Th epitope molecule (named the Gnc molecule), in silico. Subsequently, computer analysis was used to conduct a comprehensive and in-depth study of the various properties of the molecule and its effects as a vaccine molecule in the body. The Gnc molecule was designed for DNA vaccines and optimized with a lysosomal-targeting membrane protein (LAMP) strategy. The effects of GP-derived Th epitopes and multiepitope vaccines were initially verified in animals. Our research has resulted in the design of two vaccines based on effective antiviral immune targets. The effectiveness of molecular therapies has also been preliminarily demonstrated in silico and in laboratory animals, which lays a foundation for the application of a vaccines strategy in the field of antivirals.

25-Hydroxycholesterol inhibits Hantavirus infection by reprogramming cholesterol metabolism.

Hantavirus causes two types of acute diseases: hemorrhagic fever with renal syndrome and hantavirus pulmonary syndrome. It is a major health concern due to its high mortality and lack of effective treatment. Type I interferon treatment has been suggested to be effective against hantavirus when treated in advance. Interferons induce multiple interferon-stimulated genes (ISGs), whose products are highly effective at resisting and controlling viruses. A product of ISGs, the enzyme cholesterol 25-hydroxylase (CH25H), catalyzes the oxidation of cholesterol to 25-hydroxycholesterol (25HC). 25HC can inhibit multiple enveloped-virus infections, but the mechanism is largely unknown, and whether 25HC plays an important role in regulating hantavirus remains unexplored. In this study, we show that Hantaan virus (HTNV), the prototype hantavirus, induced CH25H gene in infected cells. Overexpression of CH25H and treatment with 25HC, inhibited HTNV infection, possibly by lowering 3-hydroxy-3-methyl-glutaryl coenzyme A reductase (HMG-CoA reductase, HMGCR), which inhibits cholesterol biosynthesis. In addition, cholesterol-lowering drugs such as HMGCR-targeting statins have potent hantavirus inhibitory effects. Our results indicate that 25HC and some statins are potential antiviral agents effective against hantavirus infections. This study provides evidence that targeting cholesterol metabolism is promising in developing specific hantavirus antivirals and indicates the possibility of repurposing FDA-approved cholesterol-lowering drug, statins for treating hantavirus infection.

Humoral regulation of iron metabolism by extracellular vesicles drives antibacterial response.

Immediate restriction of iron initiated by the host is a critical process to protect against bacterial infections and has been described in the liver and spleen, but it remains unclear whether this response also entails a humoral mechanism that would enable systemic sequestering of iron upon infection. Here we show that upon bacterial invasion, host macrophages immediately release extracellular vesicles (EVs) that capture circulating iron-containing proteins. Mechanistically, in a sepsis model in female mice, Salmonella enterica subsp. enterica serovar Typhimurium induces endoplasmic reticulum stress in macrophages and activates inositol-requiring enzyme 1α signaling, triggering lysosomal dysfunction and thereby promoting the release of EVs, which bear multiple receptors required for iron uptake. By binding to circulating iron-containing proteins, these EVs prevent bacteria from iron acquisition, which inhibits their growth and ultimately protects against infection and related tissue damage. Our findings reveal a humoral mechanism that can promptly regulate systemic iron metabolism during bacterial infection.

Construction and evaluation of DNA vaccine encoding Crimean Congo hemorrhagic fever virus nucleocapsid protein, glycoprotein N-terminal and C-terminal fused with LAMP1.

Crimean-Congo hemorrhagic fever virus (CCHFV) can cause severe hemorrhagic fever in humans and is mainly transmitted by ticks. There is no effective vaccine for Crimean-Congo hemorrhagic fever (CCHF) at present. We developed three DNA vaccines encoding CCHFV nucleocapsid protein (NP), glycoprotein N-terminal (Gn) and C-terminal (Gc) fused with lysosome-associated membrane protein 1 (LAMP1) and assessed their immunogenicity and protective efficacy in a human MHC (HLA-A11/DR1) transgenic mouse model. The mice that were vaccinated three times with pVAX-LAMP1-CCHFV-NP induced balanced Th1 and Th2 responses and could most effectively protect mice from CCHFV transcription and entry-competent virus-like particles (tecVLPs) infection. The mice vaccinated with pVAX-LAMP1-CCHFV-Gc mainly elicited specific anti-Gc and neutralizing antibodies and provided a certain protection from CCHFV tecVLPs infection, but the protective efficacy was less than that of pVAX-LAMP1-CCHFV-NP. The mice vaccinated with pVAX-LAMP1-CCHFV-Gn only elicited specific anti-Gn antibodies and could not provide sufficient protection from CCHFV tecVLPs infection. These results suggest that pVAX-LAMP1-CCHFV-NP would be a potential and powerful candidate vaccine for CCHFV.

Increased blood CD226- inflammatory monocytes with low antigen presenting potential correlate positively with severity of hemorrhagic fever with renal syndrome.

BACKGROUND: Hantaan virus (HTNV) infection can cause severe hemorrhagic fever with renal syndrome (HFRS). Inflammatory monocytes (iMOs) are involved in early antiviral responses. Previous studies have found that blood iMOs numbers increase in the acute phase of HFRS. Here, we further identified the phenotypic characteristics of iMOs in HFRS and explored whether phenotypic changes in iMOs were associated with HFRS severity. MATERIALS AND METHODS: Blood samples from 85 HFRS patients were used for phenotypic analysis of iMOs by flow cytometry. Plasma HTNV load was determined using RT-PCR. THP-1 cells overexpressing CD226 were used to investigate the effects of CD226 on HLA-DR/DP/DQ and CD80 expression. A mouse model was used to test macrophage phenotype following HTNV infection. RESULTS: The proportion of CD226- iMOs in the acute phase of HFRS was 66.83 (35.05-81.72) %, which was significantly higher than that in the convalescent phase (5.32 (1.36-13.52) %) and normal controls (7.39 (1.15-18.11) %) (p < 0.0001). In the acute phase, the proportion of CD226- iMOs increased more in patients with more severe HFRS and correlated positively with HTNV load and negatively with platelet count. Notably, CD226- iMOs expressed lower levels of HLA-DR/DP/DQ and CD80 than CD226+ iMOs, and overexpression CD226 could enhance the expression of HLA-DR/DP/DQ and CD80. In a mouse model, HTNV also induced the expansion of CD226- macrophages, with decreased expression of I-A/I-E and CD80. CONCLUSIONS: CD226- iMOs increased during HTNV infection and the decrease in CD226 hampered the expression of HLA-DR/DP/DQ and CD80, which may promote the immune escape of HTNV and exacerbate clinical symptoms.

RIPK3 promotes hantaviral replication by restricting JAK-STAT signaling without triggering necroptosis.

Hantaan virus (HTNV) is a rodent-borne virus that causes hemorrhagic fever with renal syndrome (HFRS), resulting in a high mortality rate of 15%. Interferons (IFNs) play a critical role in the anti-hantaviral immune response, and IFN pretreatment efficiently restricts HTNV infection by triggering the expression of a series of IFN-stimulated genes (ISGs) through the Janus kinase-signal transducer and activator of transcription 1 (JAK-STAT) pathway. However, the tremendous amount of IFNs produced during late infection could not restrain HTNV replication, and the mechanism remains unclear. Here, we demonstrated that receptor-interacting protein kinase 3 (RIPK3), a crucial molecule that mediates necroptosis, was activated by HTNV and contributed to hantavirus evasion of IFN responses by inhibiting STAT1 phosphorylation. RNA-seq analysis revealed the upregulation of multiple cell death-related genes after HTNV infection, with RIPK3 identified as a key modulator of viral replication. RIPK3 ablation significantly enhanced ISGs expression and restrained HTNV replication, without affecting the expression of pattern recognition receptors (PRRs) or the production of type I IFNs. Conversely, exogenously expressed RIPK3 compromised the host's antiviral response and facilitated HTNV replication. RIPK3-/- mice also maintained a robust ability to clear HTNV with enhanced innate immune responses. Mechanistically, we found that RIPK3 could bind STAT1 and inhibit STAT1 phosphorylation dependent on the protein kinase domain (PKD) of RIPK3 but not its kinase activity. Overall, these observations demonstrated a noncanonical function of RIPK3 during viral infection and have elucidated a novel host innate immunity evasion strategy utilized by HTNV.

STING strengthens host anti-hantaviral immunity through an interferon-independent pathway.

Hantaan virus (HTNV), the prototype virus of hantavirus, could escape innate immunity by restraining type I interferon (IFN) responses. It is largely unknown whether there existed other efficient anti-hantaviral tactics in host cells. Here, we demonstrate that the stimulator of interferon genes (STING) strengthens the host IFN-independent anti-hantaviral immunity. HTNV infection activates RIG-I through IRE1-XBP 1-mediated ER stress, which further facilitates the subcellular translocation and activation of STING. During this process, STING triggers cellular autophagy by interacting with Rab7A, thus restricting viral replication. To note, the anti-hantaviral effects of STING are independent of canonical IFN signaling. Additionally, neither application of the pharmacological antagonist nor the agonist targeting STING could improve the outcomes of nude mice post HTNV challenge in vivo. However, the administration of plasmids exogenously expressing the mutant C-terminal tail (ΔCTT) STING, which would not trigger the type I IFN responses, protected the nude mice from lethal HTNV infection. In summary, our research revealed a novel antiviral pathway through the RIG-I-STING-autophagy pathway, which offered novel therapeutic strategies against hantavirus infection.

Dihydropyridine-derived calcium channel blocker as a promising anti-hantavirus entry inhibitor.

Hantaviruses, the causative agent for two types of hemorrhagic fevers, hemorrhagic fever with renal syndrome (HFRS) and hantavirus pulmonary syndrome (HPS), are distributed from Eurasia to America. HFRS and HPS have mortality rates of up to 15% or 45%, respectively. Currently, no certified therapeutic has been licensed to treat hantavirus infection. In this study, we discovered that benidipine hydrochloride, a calcium channel blocker, inhibits the entry of hantaviruses in vitro. Moreover, an array of calcium channel inhibitors, such as cilnidipine, felodipine, amlodipine, manidipine, nicardipine, and nisoldipine, exhibit similar antiviral properties. Using pseudotyped vesicular stomatitis viruses harboring the different hantavirus glycoproteins, we demonstrate that benidipine hydrochloride inhibits the infection by both HFRS- and HPS-causing hantaviruses. The results of our study indicate the possibility of repurposing FDA-approved calcium channel blockers for the treatment of hantavirus infection, and they also indicate the need for further research in vivo.