Interferon Induction Research Articles

Type I Interferon Signalling and Ischemic Stroke: Mechanisms and Therapeutic Potentials.

Type I interferon (IFN-I) signalling is intricately involved in the pathogenesis of multiple infectious diseases, autoimmune diseases, and neurological diseases. Acute ischemic stroke provokes overactivation of IFN-I signalling within the injured brain, particularly in microglia. Following cerebral ischemia, damage-associated molecular patterns (DAMPs) released from injured neural cells elicit marked proinflammatory episodes within minutes. Among these, self-nucleic acids, including nuclear DNA and mitochondrial DNA (mtDNA), have been recognized as a critical alarm signal to fan the flames of neuroinflammation, predominantly via inducing IFN-I signalling activation in microglia. The concept of interferon-responsive microglia (IRM), marked by upregulation of a plethora of IFN-stimulated genes, has been emergingly elucidated in ischemic mouse brains, particularly in aged ones. Among the pattern recognition receptors responsible for IFN-I induction, cyclic GMP-AMP synthase (cGAS)-stimulator of interferon genes (STING) plays integral roles in potentiating microglia-driven neuroinflammation and secondary brain injury after cerebral ischemia. Here, we aim to provide an up-to-date review on the multifaceted roles of IFN-I signalling, the detailed molecular and cellular mechanisms leading to and resulting from aberrant IFN-I signalling activation after cerebral ischemia, and the therapeutic potentials. A thorough exploration of these above points will inform our quest for IFN-based therapies as effective immunomodulatory therapeutics to complement the limited repertoire of thrombolytic agents, thereby facilitating the translation from bench to bedside.

Inhibition of Aurora Kinase Induces Endogenous Retroelements to Induce a Type I/III IFN Response via RIG-I.

Some cancers deactivate STING signaling to avoid consequences of DNA damage from aberrant cell division. The surprising activation of MAVS/RIG-I signaling by AURKi might represent a vulnerability in STING signaling deficient cancers.

STING activator 2'3'-cGAMP enhanced HSV-1-based oncolytic viral therapy.

Oncolytic viruses (OVs) can selectively replicate in tumor cells and remodel the microenvironment of immunologically cold tumors, making them a promising strategy to evoke antitumor immunity. Similarly, agonists of the stimulator of interferon genes (STING)-interferon (IFN) pathway, the main cellular antiviral system, provide antitumor benefits by inducing the activation of dendritic cells (DC). Considering how the activation of the STING-IFN pathway could potentially inhibit OV replication, the use of STING agonists alongside OV therapy remains largely unexplored. Here, we explored the antitumor efficacy of combining an HSV-1-based OV, C-REV, with a membrane-impermeable STING agonist, 2'3'-GAMP. Our results demonstrated that tumor cells harbor a largely defective STING-IFN pathway, thereby preventing significant antiviral IFN induction regardless of the permeability of the STING agonist. In vivo, the combination therapy induced more proliferative KLRG1-high PD1-low CD8+ T-cells and activated CD103+ DC in the tumor site and increased tumor-specific CD44+ CD8+ T-cells in the lymph node. Overall, the combination therapy of C-REV with 2'3'-cGAMP elicited antitumor immune memory responses and significantly enhanced systemic antitumor immunity in both treated and non-treated distal tumors.

Type I IFN in Glomerular Disease: Scarring beyond the STING.

The field of nephrology has recently directed a considerable amount of attention towards the stimulator of interferon genes (STING) molecule since it appears to be a potent driver of chronic kidney disease (CKD). STING and its activator, the cyclic GMP-AMP synthase (cGAS), along with intracellular RIG-like receptors (RLRs) and toll-like receptors (TLRs), are potent inducers of type I interferon (IFN-I) expression. These cytokines have been long recognized as part of the mechanism used by the innate immune system to battle viral infections; however, their involvement in sterile inflammation remains unclear. Mounting evidence pointing to the involvement of the IFN-I pathway in sterile kidney inflammation provides potential insights into the complex interplay between the innate immune system and damage to the most sensitive segment of the nephron, the glomerulus. The STING pathway is often cited as one cause of renal disease not attributed to viral infections. Instead, this pathway can recognize and signal in response to host-derived nucleic acids, which are also recognized by RLRs and TLRs. It is still unclear, however, whether the development of renal diseases depends on subsequent IFN-I induction or other processes involved. This review aims to explore the main endogenous inducers of IFN-I in glomerular cells, to discuss what effects autocrine and paracrine signaling have on IFN-I induction, and to identify the pathways that are implicated in the development of glomerular damage.

P. gingivalis–Induced TLR2 Interactome Analysis Reveals Association with PARP9

Porphyromonas gingivalis is a Gram-negative anaerobic bacterium strongly associated with periodontal disease. Toll-like receptor 2 (TLR2) is indispensable for the host response to P. gingivalis, but P. gingivalis escapes from immune clearance via TLR2-dependent activation of phosphoinositide-3-kinase (PI3K). To probe the TLR2-dependent escape pathway of P. gingivalis, we analyzed the TLR2 interactome induced following P. gingivalis infection or activation by a synthetic lipopeptide TLR2/1 agonist on human macrophages overexpressing TLR2. Interacting proteins were stabilized by cross-linking and then immunoprecipitated and analyzed by mass spectrometry. In total, 792 proteins were recovered and network analysis enabled mapping of the TLR2 interactome at baseline and in response to infection. The P. gingivalis infection-induced TLR2 interactome included the poly (ADP-ribose) polymerase family member mono-ADP-ribosyltransferase protein 9 (PARP9) and additional members of the PARP9 complex (DTX3L and NMI). PARP9 and its complex members are highly upregulated in macrophages exposed to P. gingivalis or to the synthetic TLR2/1 ligand Pam3Cys-Ser-(Lys)4 (PAM). Consistent with its known role in virally induced interferon production, PARP9 knockdown blocked type I interferon (IFN-I) production in response to P. gingivalis and reduced inflammatory cytokine production. We found that P. gingivalis drives signal transducer and activation of transcription (STAT) 1 (S727) phosphorylation through TLR2-PARP9, explaining PARP9’s role in the induction of IFN-I downstream of TLR2. Furthermore, PARP9 knockdown reduced PI3K activation by P. gingivalis, leading to improved macrophage bactericidal activity. In summary, PARP9 is a novel TLR2 interacting partner that enables IFN-I induction and P. gingivalis immune escape in macrophages downstream of TLR2 sensing.

CGAS-STING-mediated novel nonclassic antiviral activities.

Stimulatorof interferon genes (STING) is an intracellular sensor of cyclic dinucleotides involved in the innate immune response against pathogen- or self-derived DNA. For years, interferon (IFN) induction of cyclic GMP-AMP synthase (cGAS)-STING has been considered as a canonical pattern defending the host from viral invasion. The mechanism of the cGAS-STING-IFN pathway has been well-illustrated. However, other signalling cascades driven by cGAS-STING have emerged in recent years and some of them have been found to possess antiviral ability independent of IFN. Here, we summarize the current progress on cGAS-STING-mediated nonclassic antiviral activities with an emphasis on the nuclear factor-κB and autophagy pathways, which are the most-studied pathways. In addition, we briefly present the primordial function of the cGAS-STING pathway in primitive species to show the importance of IFN-unrelated antiviral activity from an evolutionary angle. Finally, we discuss open questions that need to be solved for further exploitation of this field.

Ebola virus sequesters IRF3 in viral inclusion bodies to evade host antiviral immunity.

Viral inclusion bodies (IBs) commonly form during the replication of Ebola virus (EBOV) in infected cells, but their role in viral immune evasion has rarely been explored. Here, we found that interferon regulatory factor 3 (IRF3), but not TANK-binding kinase 1 (TBK1) or IκB kinase epsilon (IKKε), was recruited and sequestered in viral IBs when the cells were infected by EBOV transcription- and replication-competent virus-like particles (trVLPs). Nucleoprotein/virion protein 35 (VP35)-induced IBs formation was critical for IRF3 recruitment and sequestration, probably through interaction with STING. Consequently, the association of TBK1 and IRF3, which plays a vital role in type I interferon (IFN-I) induction, was blocked by EBOV trVLPs infection. Additionally, IRF3 phosphorylation and nuclear translocation induced by Sendai virus or poly(I:C) stimulation were suppressed by EBOV trVLPs. Furthermore, downregulation of STING significantly attenuated VP35-induced IRF3 accumulation in IBs. Coexpression of the viral proteins by which IB-like structures formed was much more potent in antagonizing IFN-I than expression of the IFN-I antagonist VP35 alone. These results suggested a novel immune evasion mechanism by which EBOV evades host innate immunity.

Ebola virus sequesters IRF3 in viral inclusion bodies to evade host antiviral immunity

Viral inclusion bodies (IBs) commonly form during the replication of Ebola virus (EBOV) in infected cells, but their role in viral immune evasion has rarely been explored. Here, we found that interferon regulatory factor 3 (IRF3), but not TANK-binding kinase 1 (TBK1) or IκB kinase epsilon (IKKε), was recruited and sequestered in viral IBs when the cells were infected by EBOV transcription- and replication-competent virus-like particles (trVLPs). Nucleoprotein/virion protein 35 (VP35)-induced IBs formation was critical for IRF3 recruitment and sequestration, probably through interaction with STING. Consequently, the association of TBK1 and IRF3, which plays a vital role in type I interferon (IFN-I) induction, was blocked by EBOV trVLPs infection. Additionally, IRF3 phosphorylation and nuclear translocation induced by Sendai virus or poly(I:C) stimulation were suppressed by EBOV trVLPs. Furthermore, downregulation of STING significantly attenuated VP35-induced IRF3 accumulation in IBs. Coexpression of the viral proteins by which IB-like structures formed was much more potent in antagonizing IFN-I than expression of the IFN-I antagonist VP35 alone. These results suggested a novel immune evasion mechanism by which EBOV evades host innate immunity.

In Vitro and In Vivo Evaluation of the Pathology and Safety Aspects of Three- and Four-Way Junction RNA Nanoparticles.

RNA therapeutics has advanced into the third milestone in pharmaceutical drug development, following chemical and protein therapeutics. RNA itself can serve as therapeutics, carriers, regulators, or substrates in drug development. Due to RNA's motile, dynamic, and deformable properties, RNA nanoparticles have demonstrated spontaneous targeting and accumulation in cancer vasculature and fast excretion through the kidney glomerulus to urine to prevent possible interactions with healthy organs. Furthermore, the negatively charged phosphate backbone of RNA results in general repulsion from negatively charged lipid cell membranes for further avoidance of vital organs. Thus, RNA nanoparticles can spontaneously enrich tumor vasculature and efficiently enter tumor cells via specific targeting, while those not entering the tumor tissue will clear from the body quickly. These favorable parameters have led to the expectation that RNA has low or little toxicity. RNA nanoparticles have been well characterized for their anticancer efficacy; however, little detail on RNA nanoparticle pathology and safety is known. Here, we report the in vitro and in vivo assessment of the pathology and safety aspects of different RNA nanoparticles including RNA three-way junction (3WJ) harboring 2'-F modified pyrimidine, folic acid, and Survivin siRNA, as well as the RNA four-way junction (4WJ) harboring 2'-F modified pyrimidine and 24 copies of SN38. Both animal models and patient serum were investigated. In vitro studies include hemolysis, platelet aggregation, complement activation, plasma coagulation, and interferon induction. In vivo studies include hematoxylin and eosin (H&E) staining, hematological and biochemical analysis as the serum profiling, and animal organ weight study. No significant toxicity, side effect, or immune responses were detected during the extensive safety evaluations of RNA nanoparticles. These results further complement previous cancer inhibition studies and demonstrate RNA nanoparticles as an effective and safe drug delivery vehicle for future clinical translations.

Vinorelbine causes a neuropathic pain-like state in mice via STING and MNK1 signaling associated with type I interferon induction

Type I interferons (IFNs) increase the excitability of dorsal root ganglia (DRGs) neurons via MNK-eIF4E signaling to promote pain sensitization in mice. Activation of stimulator of interferon response cGAMP interactor 1 (STING) signaling is pivotal for type I IFN induction. We hypothesized that vinorelbine, a chemotherapeutic and activator of STING, would cause a neuropathic pain-like state in mice via STING signaling in DRG neurons associated with IFN production. Vinorelbine caused tactile allodynia and grimacing in wild-type (WT) mice and increased p-IRF3, type I IFNs, and p-eIF4E in peripheral nerves. Supporting our hypothesis, vinorelbine failed to induce IRF3-IFNs-MNK-eIF4E in StingGt/Gt mice and, subsequently, failed to cause pain. The vinorelbine-elicited increase of p-eIF4E was not observed in Mknk1-/- (MNK1 knockout) mice in peripheral nerves consistent with the attenuated pro-nociceptive effect of vinorelbine in these mice. Our findings show that activation of STING signaling in the periphery causes a neuropathic pain-like state through type I IFN signaling to DRG nociceptors.

New immunomodulatory preparations based on RNA to treat viral infections

New experimental antiviral preparations are developed based on a molecular structure consisting of single-stranded and double-stranded RNAs from different sources and an outer envelope based on dextran with specific isotropic agents. An affection of developed molecular structure preparations on physiological indicators variety in laboratory animals to obtain data on the tolerance of compositions was investigated. It has been shown that administering higher doses of experimental preparations to animals doesn`t cause or lead to short-term and minor deviations in their condition. An application of the method of specific interferon-inducing activity determining in the study of antiviral and other interferon inducers characteristics may be used for estimation of criteria like the minimal effective interferon-inducing dose (MEID) and the modified chemotherapeutical index (mCTI), required for a complete characterisation of drugs.

The Asthma Risk Gene, GSDMB, Promotes Mitochondrial DNA-induced ISGs Expression.

Released mitochondrial DNA (mtDNA) in cells activates cGAS-STING pathway, which induces expression of interferon-stimulated genes (ISGs) and thereby promotes inflammation, as frequently seen in asthmatic airways. However, whether the genetic determinant, Gasdermin B (GSDMB), the most replicated asthma risk gene, regulates this pathway remains unknown. We set out to determine whether and how GSDMB regulates mtDNA-activated cGAS-STING pathway and subsequent ISGs induction in human airway epithelial cells. Using qPCR, ELISA, native polyacrylamide gel electrophoresis, co-immunoprecipitation and immunofluorescence assays, we evaluated the regulation of GSDMB on cGAS-STING pathway in both BEAS-2B cells and primary normal human bronchial epithelial cells (nHBEs). mtDNA was extracted in plasma samples from human asthmatics and the correlation between mtDNA levels and eosinophil counts was analyzed. GSDMB is significantly associated with RANTES expression in asthmatic nasal epithelial brushing samples from the Genes-environments and Admixture in Latino Americans (GALA) II study. Over-expression of GSDMB promotes DNA-induced IFN and ISGs expression in bronchial epithelial BEAS-2B cells and nHBEs. Conversely, knockout of GSDMB led to weakened induction of interferon (IFNs) and ISGs in BEAS-2B cells. Mechanistically, GSDMB interacts with the C-terminus of STING, promoting the translocation of STING to Golgi, leading to the phosphorylation of IRF3 and induction of IFNs and ISGs. mtDNA copy number in serum from asthmatics was significantly correlated with blood eosinophil counts especially in male subjects. GSDMB promotes the activation of mtDNA and poly (dA:dT)-induced activation of cGAS-STING pathway in airway epithelial cells, leading to enhanced induction of ISGs.

Multi-omics analysis reveals the impact of influenza a virus host adaptation on immune signatures in pig tracheal tissue.

Influenza A virus (IAV) infection is a global respiratory disease, which annually leads to 3-5 million cases of severe illness, resulting in 290,000-650,000 deaths. Additionally, during the past century, four global IAV pandemics have claimed millions of human lives. The epithelial lining of the trachea plays a vital role during IAV infection, both as point of viral entry and replication as well as in the antiviral immune response. Tracheal tissue is generally inaccessible from human patients, which makes animal models crucial for the study of the tracheal host immune response. In this study, pigs were inoculated with swine- or human-adapted H1N1 IAV to gain insight into how host adaptation of IAV shapes the innate immune response during infection. In-depth multi-omics analysis (global proteomics and RNA sequencing) of the host response in upper and lower tracheal tissue was conducted, and results were validated by microfluidic qPCR. Additionally, a subset of samples was selected for histopathological examination. A classical innate antiviral immune response was induced in both upper and lower trachea after infection with either swine- or human-adapted IAV with upregulation of genes and higher abundance of proteins associated with viral infection and recognition, accompanied by a significant induction of interferon stimulated genes with corresponding higher proteins concentrations. Infection with the swine-adapted virus induced a much stronger immune response compared to infection with a human-adapted IAV strain in the lower trachea, which could be a consequence of a higher viral load and a higher degree of inflammation. Central components of the JAK-STAT pathway, apoptosis, pyrimidine metabolism, and the cytoskeleton were significantly altered depending on infection with swine- or human-adapted virus and might be relevant mechanisms in relation to antiviral immunity against putative zoonotic IAV. Based on our findings, we hypothesize that during host adaptation, IAV evolve to modulate important host cell elements to favor viral infectivity and replication.

Synthesis and biological evaluation of lipid A derived from commensal Bacteroides.

The inflammation-inducing properties of lipopolysaccharides (LPS) of Gram-negative bacteria reside in their lipid A moiety. Bacillus fragilis, which is a commensal Gram-negative bacterium, biosynthesises lipid A that is structurally distinct from that of E. coli and other enteric bacteria. It is composed of a β1,6-linked glucosamine (GlcN) disaccharide that is only phosphorylated at the anomeric center. The major species of B. fragilis has five fatty acids and the amine of the distal GlcN moiety carries the unusual (R)-3-(13-methyltetradecanoyloxy)-1.5-methylhexadecanoic acid. A recent study indicates that the LPS of B. fragilis has anti-viral activity by selective induction of interferon (IFN)-β and is protective in mouse models of vesicular stomatitis virus (VSV) and influenza A. Heterogeneity in the structures of LPS and lipid A and possible contamination with other inflammatory components make it difficult to unambiguously define the immune-modulatory properties of LPS or lipid A. Therefore, we developed a synthetic approach for the preparation of the unusual major lipid A species derived from B. fragilis, which includes a synthetic approach for (R)-3-(13-methyltetradecanoyloxy)-1.5-methylhexadecanoic acid by the Wittig olefination to install the terminal isopropyl moiety. The proinflammatory and antiviral responses of synthetic B. fragilis lipid A were investigated in several cell lines and primary human monocytes by examining the production of interleukin (IL)-6 and IFN-β. It was found that B. fragilis does not induce the production of IL-6 and IFN-β but can partially antagonize the production of pro-inflammatory cytokines induced by E. coli LPS and lipid A.

Recurrent herpetic erosion of the cornea: diagnosis, treatment and prevention of recurrences

A group of patients was found to have a special form of recurrent corneal erosion caused by types I and II herpes virus. This form represents an independent form of ophthalmic herpes - herpetic recurrent erosion (HRE) of the cornea. The herpetic etiology of recurrent corneal erosion was confirmed by the immunofluorescence study of scraping from the conjunctiva, which revealed a high concentration of the herpes simplex virus antigen. Treatment of patients (171 patients, 182 eyes) with HRE included 2 consecutive stages: stage I - relief of acute symptoms of the disease with the help of conservative treatment (instillations of interferon inducers, autologous serum, corneal protectors, tear substitutes, use of therapeutic soft contact lenses); in some cases, phototherapeutic keratectomy was used in the absence of the effect of conservative therapy, as well as in the localization of the focus in the optical zone. Stage II involved anti-relapse therapy based on the use of a Russian-produced herpes vaccine in the intercurrent period. After vaccination, observation for 2 years or more showed that 81.3% of patients achieved clinical recovery (complete cessation of HRE recurrences), 15.8% had a decrease in the frequency and severity of relapses, while 2.9% of patients did not respond to the treatment.

Antiviral Activity of Double-Stranded Ribonucleic Acid and Interferon Alpha Composition in the Model of Experimental Influenza Infection of Mice

High variability of influenza viruses requires the development of agents for nonspecific resistance stimulation, along with the development of new drugs for prevention and treatment. Among the antiviral drugs, interferons and their inducers are known to exhibit a universally wide spectrum of action. The Institute of Medical Biotechnology, a branch of SRC VB «Vector», Rospotrebnadzor, has developed the technology and obtained pharmaceutical compositions containing an interferon inducer — double stranded ribonucleic acid (dsRNA) from the killer strain of Saccharomyces cerevisiae yeast and recombinant human interferon-alpha-2b (IFN- alpha-2b). The specific antiviral activity of the preparations and synergistic effect of the components within the compositions were shown in L-68 and L-929 cell cultures. The aim of this work was to study antiviral activity of intranasal forms of the pharmaceutical compositions containing yeast dsRNA and recombinant human interferon-alpha-2b in a model of lethal influenza infection in mice. The outbred ICR/CD1 mice were intranasally infected with influenza A/Aichi/2/68 (H3N2) virus. The study compositions were intranasally administered 3 hours before infection with influenza virus, as well as 1 and 3 days post infection. The doses of active components in the administered compositions were as follows: for dsRNA — 2.5 mg/kg, for IFN-alpha-2b — 500 IU/kg, 2500 IU/kg, or 5000 IU/kg. The antiviral activity of the drugs was assessed based on the mortality rate and the average life expectancy of mice. It was shown that a three-time intranasal administration of the composition of dsRNA (2.5 mg/kg) and IFN-alpha-2b (2500 IU) into the infected mice according to therapeutic-prophylactic regimen led to an increase in the rates of survival and average life expectancy of animals, which were comparable to the effect of Tamiflu. The comparison preparations — dsRNA and IFN-alpha-2b — administered intranasally at the same doses and regimen exerted no antiviral effect in this mouse model of viral infection. The data obtained confirm the prospects for further development of new dosage forms of dsRNA and interferons for intranasal application as agents for prevention and treatment of influenza.

Factor inhibiting HIF negatively regulates antiviral innate immunity via hydroxylation of IKKϵ

Activation of type I interferon (IFN-1) signaling is essential to protect host cells from viral infection. The full spectrum of IFN-I induction requires the activation of a number of cellular factors, including IκB kinase epsilon (IKKϵ). However, the regulation of IKKϵ activation in response to viral infection remains largely unknown. Here, we show that factor inhibiting hypoxia-inducible factor (HIF) (FIH), an asparaginyl hydroxylase, interacts with IKKϵ and catalyzes asparagine hydroxylation of IKKϵ at Asn-254, Asn-700, and Asn-701, resulting in the suppression of IKKϵ activation. FIH-mediated hydroxylation of IKKϵ prevents IKKϵ binding to TBK1 and TRAF3 and attenuates the cIAP1/cIAP2/TRAF2 E3 ubiquitin ligase complex-catalyzed K63-linked polyubiquitination of IKKϵ at Lys-416. In addition, Fih-deficient mice and zebrafish are more resistant to viral infection. This work uncovers a previously unrecognized role of FIH in suppressing IKKϵ activation for IFN signaling and antiviral immune responses.

Efficacy of interferon inducers against Chikungunya virus <i>in vitro</i>

Scientific relevance. To date, no specific antivirals have been approved to treat and prevent Chikungunya fever, its complications, and sequelae. Therefore, the development of therapeutic and preventive medicinal products against Chikungunya virus (CHIKV), including interferon inducers, is gaining relevance.Aim. The authors aimed to study the effectiveness of prophylactic administration of an interferon inducer against CHIKV in an in vitro model.Materials and methods. The study used two cell lines (Vero and А549), a CHIKV strain (Nika2021), and an interferon-inducing medicinal product (double-stranded RNA sodium salt) at two doses (250 μg/mL and 500 μg/mL) administered at two schedules: Prevention (4 h prior to the virus challenge) and Emergency Prevention (at the time of the virus challenge). The authors determined the CHIKV titre by its cytopathogenic effect, the CHIKV RNA content by the cycle threshold value in real-time reverse-transcription polymerase chain reaction, and the concentration of cytokines using the enzyme immunoassay method. The study monitored the changes in CHIKV biological activity, CHIKV RNA levels, and the production of interferon-alpha (IFN-α), interferon-gamma (IFN-γ), interleukin-6 (IL-6), and tumour necrosis factor-alpha (TNF-α) in cells over time. The statistical analysis of the resulting data used Microsoft Office Excel 2016 and StatTech.Results. The medicinal product at doses of 250 μg/mL and 500 μg/mL stimulated the production of both IFN-α and IFN-γ (IFN-α to a greater extent than IFN-γ) in both cell lines (in A549 to a greater extent than in Vero). The changes in CHIKV RNA levels with time corresponded to those of the virus titre. In general, CHIKV RNA levels in Vero cells were significantly higher than those in A549 cells (р<0.002 at 250 μg/mL and р<0.0005 at 500 μg/mL). The CHIKV RNA content after preventive interferon inducer administration was significantly lower than that in the control experiment (challenge without administration of the medicinal product) for both doses and both cell lines (р<0.002 for Vero cells; р<0.0003 for А549 cells). The CHIKV RNA content after interferon inducer administration as emergency prevention was significantly lower than that in the control experiment (р<0.05 for Vero cells; р<0.003 for А549 cells). The study demonstrated the efficacy of the interferon inducer against CHIKV and a higher applicability of the A549 cell line to studying antiviral activity in vitro. The authors observed the production of IL-6 and TNF-α by intact cells of both lines.Conclusions. According to the results, the studied interferon inducer has a positive antiviral effect against CHIKV in vitro, with the antiviral effect degree depending on the cell line used. This experimental study demonstrated the need to carefully select the cell line for a study in accordance with its objectives and to evaluate the production of cytokines by a monolayer of cells before stimulation with viruses and/or medicinal products.

Virus-Host Protein Interaction Network of the Hepatitis E Virus ORF2-4 by Mammalian Two-Hybrid Assays.

Throughout their life cycle, viruses interact with cellular host factors, thereby influencing propagation, host range, cell tropism and pathogenesis. The hepatitis E virus (HEV) is an underestimated RNA virus in which knowledge of the virus-host interaction network to date is limited. Here, two related high-throughput mammalian two-hybrid approaches (MAPPIT and KISS) were used to screen for HEV-interacting host proteins. Promising hits were examined on protein function, involved pathway(s), and their relation to other viruses. We identified 37 ORF2 hits, 187 for ORF3 and 91 for ORF4. Several hits had functions in the life cycle of distinct viruses. We focused on SHARPIN and RNF5 as candidate hits for ORF3, as they are involved in the RLR-MAVS pathway and interferon (IFN) induction during viral infections. Knocking out (KO) SHARPIN and RNF5 resulted in a different IFN response upon ORF3 transfection, compared to wild-type cells. Moreover, infection was increased in SHARPIN KO cells and decreased in RNF5 KO cells. In conclusion, MAPPIT and KISS are valuable tools to study virus-host interactions, providing insights into the poorly understood HEV life cycle. We further provide evidence for two identified hits as new host factors in the HEV life cycle.

TBK1 upregulates the interferon response against virus by the TBK1-IRF3/7 axis in yellow catfish (Pelteobagrus fulvidraco)

Yellow catfish (Pelteobagrus fulvidraco) is an important economic species of freshwater fish, widely distributed in China. Recently, viral diseases of yellow catfish have been identified in Chian (Hubei province), arising more attention to the viral immunity in P. fulvidraco. Tumor necrosis factor (TNF) receptor-associated factor NF-κB activator (TANK)-binding kinase 1 (TBK1) plays an essential role in IFN production and innate antiviral immunity. In the present study, we characterized the P. fulvidraco TBK1 (PfTBK1) and reported its function in interferon response. The full-length open reading frame (ORF) is 2184 bp encoding a protein with 727 amino acids, which is composed of four conserved domains, including KD, ULD, CCD1, and CCD2, similar to TBK1 in other species. Pftbk1 was widely expressed in all detected tissues by qPCR and was not inducible by the spring viremia of carp virus (SVCV), a single-strand RNA virus. In addition, the cellular distribution indicated that PfTBK1 was only located in the cytoplasm. Moreover, PfTBK1 induced strong IFN promoter activities through the Jak-stat pathway, and PfTBK1 interacted with and significantly phosphorylated IFN regulatory factor 3/7 (IRF3/7) in P. fulvidraco, promoting the nuclear translocation of pfIRF3 and PfIRF7, and PfTBK1 upregulated IFN response by PfTBK1-PfIRF3/7 axis. Above all, PfTBK1 triggered IFN response and strongly inhibited the replication of SVCV in EPC cells through induction of IFN downstream IFN-stimulated genes (ISGs). Summarily, this work reveals that PfTBK1 plays a positive regulatory role in IFN induction through the TBK1-IRF3/7 axis, laying a foundation for further exploring the molecular mechanism of the antiviral process in P. fulvidraco.