Necroptosis blockade prevents lung injury in severe influenza.

Severe influenza A virus (IAV) infections can result in hyper-inflammation, lung injury and acute respiratory distress syndrome1-5 (ARDS), for which there are no effective pharmacological therapies. Necroptosis is an attractive entry point for therapeutic intervention in ARDS and related inflammatory conditions because it drives pathogenic lung inflammation and lethality during severe IAV infection6-8 and can potentially be targeted by receptor interacting protein kinase 3 (RIPK3) inhibitors. Here we show that a newly developed RIPK3 inhibitor, UH15-38, potently and selectively blocked IAV-triggered necroptosis in alveolar epithelial cells in vivo. UH15-38 ameliorated lung inflammation and prevented mortality following infection with laboratory-adapted and pandemic strains of IAV, without compromising antiviral adaptive immune responses or impeding viral clearance. UH15-38 displayed robust therapeutic efficacy even when administered late in the course of infection, suggesting that RIPK3 blockade may provide clinical benefit in patients with IAV-driven ARDS and other hyper-inflammatory pathologies.

Inhibition of influenza A virus replication by a marine derived quinolone alkaloid targeting virus nucleoprotein.

Owing to the emergence of drug resistance and high morbidity and mortality, the need for novel anti-influenza A virus (IAV) drugs with divergent targets is highly sought after. Herein, a novel quinolone alkaloid (QLA) derived from marine fungus was discovered with broad-spectrum anti-IAV activities with low toxicity. Distinct from current anti-IAV drugs, QLA may block virus replication and viral RNA (vRNA) export from the nucleus by targeting virus nucleoprotein (NP). QLA can block the binding of chromosome region maintenance 1 to nuclear export signal 3 of NP to inhibit the nuclear export of NP and vRNP. QLA may also affect vRNP assembly by interfering with the binding of NP to RNA rather than NP oligomerization. Arg305 and Phe488-Gly490 may be required for the interaction between QLA and NP, and the binding pocket around these amino acids may be a promising target for anti-IAV drugs. Importantly, oral administration of QLA can protect the mice against IAV-induced death and weight loss, superior to the effects of the clinical drug oseltamivir. In summary, the marine derived compound QLA has the potential to be developed into a novel anti-IAV agent targeting virus NP protein in the future.

In vitro virucidal activity of the theaflavin-concentrated tea extract TY-1 against influenza A virus.

The annual spread of influenza A virus (IAV) infection is a global concern. We examined the IAV-inactivating potential of theaflavin-concentrated tea extract TY-1, which contains abundant polyphenols, including concentrated theaflavins and catechins. TY-1 exhibited concentration- and time-dependent virucidal activity against IAV. Specifically, 5.0 mg/mL TY-1 induced a 1.33 and ≥ 5.17 log10 50% tissue culture infective dose/mL reduction of the viral titer compared with dextrin as the diluent control within 30 min and 6 h reaction time, respectively. The high virucidal activity of TY-1 was attributed to the combined additive activities of multiple virucidal components, including theaflavins, which led to an investigation of the virucidal mechanism of action of TY-1. Western blotting revealed that TY-1 treatment reduced the band intensity of hemagglutinin and induced the appearance of additional high molecular mass bands/ladders. In addition, TY-1 treatment also reduced the band intensity of neuraminidase (NA). A hemagglutination assay revealed that TY-1 reduced hemagglutination activity, and an NA assay revealed reduced NA activity. These results indicated that TY-1 caused structural abnormalities in IAV spike proteins, possibly leading to their destruction. Reverse transcription polymerase chain reaction (PCR) targeting the IAV genome and electron microscopic observation of viral particles revealed that upon application of TY-1, the PCR products dissipated, which indicates that TY-1 destroyed the IAV genome, and the number of viral particles reduced. Overall, TY-1 exhibited multiple modes of IAV-inactivating activity. Our findings support the possible future practical use of TY-1 as a virucidal supplemental agent that can contribute to IAV infection control.

Role of sialylated glycans on bovine lactoferrin against influenza virus.

Influenza is a worldwide plague caused by the influenza virus (IAV) infection, which is initiated by specific recognition with sialic acids on host cell surface. Bovine lactoferrin (bLf) is a sialoglycoprotein belonging to the transferrin family, and it plays an important role in immune regulation. It also shows toxicity against cancer cells and pathogenic microorganisms including bacteria, fungi, and virus. The purpose of this study is to assess the roles of the sialylated glycans on bLf against IAV. To this end, bLf were first treated with sodium periodate to destroy its sialylated glycans. Then, the binding activity of native or desialylated bLf with various IAV was assessed by blotting assay. Finally, their ability to inhibit IAV attachment to host cells was analyzed in vitro. Our result showed that the sialylated glycans on bLf were almost completely destroyed by sodium periodate treatment. Furthermore, the binding activity of desialylated bLf to IAV and the ability to inhibit IAV mimics binding to MDCK cells were significantly reduced compared to that of native bLf. These results demonstrated that the sialylated glycans on bLf could serve as competitive substrates to block IAV attachment to host cells during the early stages of viral infection. Our findings make an important contribute for the fully understanding of the mechanism of bLf in the prevention of IAV infections and their possible applications in antiviral infection.

Short-hairpin RNAs delivered by recombinant adeno-associated virus inhibited the replication of influenza A viruses in vitro.

Antiviral short-hairpin RNAs (shRNAs) delivered by recombinant adeno-associated virus (rAAV) were investigated for their potential prophylactic and therapeutic applications related to the influenza A virus (IAV). To express shRNAs efficiently, an H1 promoter was inserted into the commercial rAAV2 system. The modified rAAV2 system could express shRNAs, and the purified rAAV was obtained at levels over 1013 viral genomes/ml and 1010 viral infection units/ml. The shNP-1496-n and shM2-925 delivered by rAAV could inhibit the replication of the H1N1 and H5N1 virus by targeting the conserved regions of the IAV nucleoprotein and matrix 2 genes in MDCK cells. The shNP-1496-n and shM2-925 expressed by rAAV could provide potent and long-term anti-H5N1 virus effects in rAAV-shRNA-enriched MDCK cells. Our findings provide a rational basis for developing RNA interference for the prevention and therapy of IAV infection.

Anti-influenza A Virus Effects and Mechanisms of Emodin and Its Analogs via Regulating PPARα/γ-AMPK-SIRT1 Pathway and Fatty Acid Metabolism.

The peroxisome proliferator-activated receptor (PPAR) α/γ-adenosine 5'-monophosphate- (AMP-) activated protein kinase- (AMPK-) sirtuin-1 (SIRT1) pathway and fatty acid metabolism are reported to be involved in influenza A virus (IAV) replication and IAV-pneumonia. Through a cell-based peroxisome proliferator responsive element- (PPRE-) driven luciferase bioassay, we have investigated 145 examples of traditional Chinese medicines (TCMs). Several TCMs, such as Polygonum cuspidatum, Rheum officinale Baillon, and Aloe vera var. Chinensis (Haw.) Berg., were found to possess high activity. We have further detected the anti-IAV activities of emodin (EMO) and its analogs, a group of common important compounds of these TCMs. The results showed that emodin and its several analogs possess excellent anti-IAV activities. The pharmacological tests showed that emodin significantly activated PPARα/γ and AMPK, decreased fatty acid biosynthesis, and increased intracellular ATP levels. Pharmaceutical inhibitors, siRNAs for PPARα/γ and AMPKα1, and exogenous palmitate impaired the inhibition of emodin. The in vivo test also showed that emodin significantly protected mice from IAV infection and pneumonia. Pharmacological inhibitors for PPARα/γ and AMPK signal and exogenous palmitate could partially counteract the effects of emodin in vivo. In conclusion, emodin and its analogs are a group of promising anti-IAV drug precursors, and the pharmacological mechanism of emodin is linked to its ability to regulate the PPARα/γ-AMPK pathway and fatty acid metabolism.

IFN-λ1 Displays Various Levels of Antiviral Activity In Vitro in a Select Panel of RNA Viruses.

Type III interferons (lambda IFNs) are a quite new, small family of three closely related cytokines with interferon-like activity. Attention to IFN-λ is mainly focused on direct antiviral activity in which, as with IFN-α, viral genome replication is inhibited without the participation of immune system cells. The heterodimeric receptor for lambda interferons is exposed mainly on epithelial cells, which limits its possible action on other cells, thus reducing the likelihood of developing undesirable side effects compared to type I IFN. In this study, we examined the antiviral potential of exogenous human IFN-λ1 in cellular models of viral infection. To study the protective effects of IFN-λ1, three administration schemes were used: 'preventive' (pretreatment); 'preventive/therapeutic' (pre/post); and 'therapeutic' (post). Three IFN-λ1 concentrations (from 10 to 500 ng/mL) were used. We have shown that human IFN-λ1 restricts SARS-CoV-2 replication in Vero cells with all three treatment schemes. In addition, we have shown a decrease in the viral loads of CHIKV and IVA with the 'preventive' and 'preventive/therapeutic' regimes. No significant antiviral effect of IFN-λ1 against AdV was detected. Our study highlights the potential for using IFN-λ as a broad-spectrum therapeutic agent against respiratory RNA viruses.

The Indirect Antiviral Potential of Long Noncoding RNAs Encoded by IFITM Pseudogenes.

The interferon-induced transmembrane (IFITM) gene family performs multiple functions in immunity, including inhibition of virus entry into cells. The IFITM repertoire varies widely between species and consists of protein-coding genes and pseudogenes. The selective forces driving pseudogenization within gene families are rarely understood. In this issue, the human pseudogene IFITM4P is characterized as a virus-induced, long noncoding RNA that contributes to restriction of influenza A virus by regulating mRNA levels of IFITM1, IFITM2, and IFITM3.

Quinazolin-derived myeloperoxidase inhibitor suppresses influenza A virus-induced reactive oxygen species, pro-inflammatory mediators and improves cell survival.

Superoxide radicals and other reactive oxygen species (ROS) are implicated in influenza A virus-induced inflammation. In this in vitro study, we evaluated the effects of TG6-44, a novel quinazolin-derived myeloperoxidase-specific ROS inhibitor, on influenza A virus (A/X31) infection using THP-1 lung monocytic cells and freshly isolated peripheral blood mononuclear cells (PBMC). TG6-44 significantly decreased A/X31-induced ROS and virus-induced inflammatory mediators in THP-1 cells (IL-6, IFN-γ, MCP-1, TNF-α, MIP-1ß) and in human PBMC (IL-6, IL-8, TNF-α, MCP-1). Interestingly, TG6-44-treated THP-1 cells showed a decrease in percent cells expressing viral nucleoprotein, as well as a delay in translocation of viral nucleoprotein into the nucleus. Furthermore, in influenza A virus-infected cells, TG6-44 treatment led to suppression of virus-induced cell death as evidenced by decreased caspase-3 activation, decreased proportion of Annexin V+PI+ cells, and increased Bcl-2 phosphorylation. Taken together, our results demonstrate the anti-inflammatory and anti-infective effects of TG6-44.

Zinc-Embedded Polyamide Fabrics Inactivate SARS-CoV-2 and Influenza A Virus.

Influenza A viruses (IAV) and SARS-CoV-2 can spread via liquid droplets and aerosols. Face masks and other personal protective equipment (PPE) can act as barriers that prevent the spread of these viruses. However, IAV and SARS-CoV-2 are stable for hours on various materials, which makes frequent and correct disposal of these PPE important. Metal ions embedded into PPE may inactivate respiratory viruses, but confounding factors such as adsorption of viruses make measuring and optimizing the inactivation characteristics difficult. Here, we used polyamide 6.6 (PA66) fibers containing embedded zinc ions and systematically investigated if these fibers can adsorb and inactivate SARS-CoV-2 and IAV H1N1 when woven into a fabric. We found that our PA66-based fabric decreased the IAV H1N1 and SARS-CoV-2 titer by approximately 100-fold. Moreover, we found that the zinc content and the virus inactivating property of the fabric remained stable over 50 standardized washes. Overall, these results provide insights into the development of reusable PPE that offer protection against RNA virus spread.

The Biological Effects of Forsythia Leaves Containing the Cyclic AMP Phosphodiesterase 4 Inhibitor Phillyrin.

Forsythia fruit (Forsythia suspensa Vahl (Oleaceae)) is a common component of Kampo medicines for treating the common cold, influenza, and allergies. The main polyphenolic compounds in the leaves of F. suspensa are pinoresinol ß-d-glucoside, phillyrin and forsythiaside, and their levels are higher in the leaves of the plant than in the fruit. It is known that polyphenolic compounds stimulate lipid catabolism in the liver and suppress dyslipidemia, thereby attenuating diet-induced obesity and polyphenolic anti-oxidants might attenuate obesity in animals consuming high-fat diets. Recently, phillyrin was reported as a novel cyclic AMP phosphodiesterase 4 (PDE4) inhibitor derived from forsythia fruit. It was expected that the leaves of F. suspensa might display anti-obesity effects and serve as a health food material. In this review, we summarized our studies on the biological effects of forsythia leaves containing phillyrin and other polyphenolic compounds, particularly against obesity, atopic dermatitis, and influenza A virus infection, and its potential as a phytoestrogen.

Lianhuaqingwen capsule inhibits influenza-induced bacterial adhesion to respiratory epithelial cells through down-regulation of cell adhesion molecules.

ETHNOPHARMACOLOGICAL RELEVANCE: Influenza virus infection is widely believed to cause mild symptoms, but can lead to high mortality and severe disease complicated by secondary bacterial pneumonia. Traditional Chinese medicine (TCM) has been proposed as a promising agent to treat respiratory viral infections. A herbal formula Lianhuaqingwen capsule (LHQW) comprising two prescriptions: Maxing Shigan decoction and Yinqiao San, has been used clinically to treat respiratory infection with immune regulatory effects. However, little is known about the capacity of LHQW against influenza-induced secondary bacterial pneumonia. AIM OF STUDY: This study aimed to evaluate the efficacy and underlying mechanism of LHQW on influenza A virus A/PR/8/34 (PR8) secondary methicillin-resistant Staphy-lococcus aureus (MRSA) infection. METHODS: The anti-adhesion activity of LHQW against PR8-induced MRSA infection was assessed in human lung epithelial (A549) cells and the effect of LHQW on the expression of intracellular adhesion molecule 1 (ICAM-1) was detected. Also, the mRNA expression levels of inflammatory cytokines upon lipopolysaccharide (LPS) stimulation in PR8-infected A549 cells were determined. The body weight change, survivals, viral titers, colonies and the pathological parameters after LHQW treatment in severe pneumonia model have all been systematically determined. RESULTS: LHQW significantly reduced the adhesion of MRSA to PR8-infected A549 cells in a dose-dependent manner by suppressing the up-regulation of bacterial receptors. LHQW also markedly declined the overexpression of IL-6, IL-8, and TNF-α induced by LPS stimulated-A549 cells following influenza virus infection. Furthermore, the abnormal changes of lung index in dual-infection mice were relieved after administered with LHQW in preventive and therapeutic mode, but with no significantly difference (P > 0.05). LHQW could not effectively improve survival rate or prolong the survival time of mice (P > 0.05). LHQW (1000 mg/kg/d) administered prophylactically significantly decreased the lung viral titers (P < 0.05), slightly downregulated IL-6 but TNF-α, IL-1ß levels and improved lung pathological inflammation including neutrophil infiltration, necrosis, which is consistent with the expression of inflammatory factors. CONCLUSIONS: LHQW inhibited influenza-induced bacterial adhesion by down-regulating the adhesion molecules with the improvement trend on severe pneumonia, indicating that it can be used as an adjuvant medication in severe viral-bacterial pneumonia therapy rather than as a single medication.

Antiviral Bafilomycins from a Feces-Inhabiting Streptomyces sp.

A new bafilomycin derivative (1) and another seven known bafilomycins (2-8) were isolated from feces-derived Streptomyces sp. HTL16. The structure of 1 was elucidated by 1D and 2D NMR spectroscopic analysis. Biological testing demonstrated that these bafilomycins exhibited potent antiviral activities against the influenza A and SARS-CoV-2 viruses, with IC50 values in the nanomolar range, by inhibiting the activity of endosomal ATP-driven proton pumps.

Synthesis and Biopharmaceutical Applications of Sugar-Based Polymers: New Advances and Future Prospects.

The rapid rise in research interest in carbohydrate-based polymers is undoubtedly due to the nontoxic nature of such materials in an in vivo environment and the versatile roles that the polymers can play in cellular functions. Such polymers have served as therapeutic tools for drug delivery, including antigens, proteins, and genes, as well as diagnostic devices. Our focus in the first half of this Review is on synthetic methods based on ring-opening polymerization and enzyme-catalyzed polymerization, along with controlled radical polymerization. In the second half of this Review, sugar-based polymers are discussed on the basis of their remarkable success in competitive receptor binding, as multifunctional nanocarriers of targeting inhibitors for cancer treatment, in genome-editing delivery, in immunotherapy based on endogenous antibody recruitment, and in treatment of respiratory diseases, including influenza A. Particular emphasis is put on the synthesis and biopharmaceutical applications of sugar-based polymers published in the most recent 5 years. A noticeable attribute of carbohydrate-based polymers is that the sugar-receptor interactions can be facilitated by the cooperative effect of multiple sugar units. Their diversified topology and structures will drive the development of new synthetic strategies and bring about important applications, including coronavirus-related drug therapy.

Laccase-Catalyzed 1,4-Dioxane-Mediated Synthesis of Belladine N-Oxides with Anti-Influenza A Virus Activity.

Belladine N-oxides active against influenza A virus have been synthetized by a novel laccase-catalyzed 1,4-dioxane-mediated oxidation of aromatic and side-chain modified belladine derivatives. Electron paramagnetic resonance (EPR) analysis confirmed the role of 1,4-dioxane as a co-oxidant. The reaction was chemo-selective, showing a high functional-group compatibility. The novel belladine N-oxides were active against influenza A virus, involving the early stage of the virus replication life cycle.

New insights into antiviral and cytotoxic potential of quercetin and its derivatives - A biochemical perspective.

Quercetin, a potential polyphenolic which possesses several biological effects. The influenza virus polymerase basic 2 (PB2) subunit of RNA polymerase responsible for replication, degree of virus conservation and active target site for designing specific antivirals. The quercetin derivatives downloaded from PubChem were screened using PyRX software configured with Vina Wizard, targeted on cap-binding site of the PB2 of influenza viral RNA polymerase. Among the PubChem library (total 97,585,747 compounds), 410 quercetin derivatives were screened using molecular docking (affinity: <-9.0 kcal) for their drug-likeness and in vitro cytopathic effect by Sulforhodamine B (SRB) assay. Among all quercetin derivatives, quercetin 3'-glucuronide (Q3G) showed strongest binding affinity towards cap-binding site of the PB2 subunit with -9.6 kcal of binding affinity and 0.00054 mM of Ki value, while quercetin 3'-glucuronide (Q7G) was presented highest anti-influenza activity with 2.10 ± 0.05 of IC50 on influenza A/PR/8/34 virus and non-cytotoxic effect as CC50 > 100 µg/mL.

Comparative in vitro analysis of inhibition of rhinovirus and influenza virus replication by mucoactive secretolytic agents and plant extracts.

BACKGROUND: Rhinoviruses and influenza viruses cause millions of acute respiratory infections annually. Symptoms of mild acute respiratory infections are commonly treated with over-the-counter products like ambroxol, bromhexine, and N-acetyl cysteine, as well as of thyme and pelargonium extracts today. Because the direct antiviral activity of these over-the-counter products has not been studied in a systematic way, the current study aimed to compare their inhibitory effect against rhinovirus and influenza virus replication in an in vitro setting. METHODS: The cytotoxicity of ambroxol, bromhexine, and N-acetyl cysteine, as well as of thyme and pelargonium extracts was analyzed in Madin Darby canine kidney (MDCK) and HeLa Ohio cells. The antiviral effect of these over-the-counter products was compared by analyzing the dose-dependent inhibition (i) of rhinovirus A2- and B14-induced cytopathic effect in HeLa Ohio cells and (ii) of influenza virus A/Hong Kong/68 (subtype H3N2)- and A/Jena/8178/09 (subtype H1N1, pandemic)-induced cytopathic effect in MDCK cells at non-cytotoxic concentrations. To get insights into the mechanism of action of pelargonium extract against influenza virus, we performed time-of-addition assays as well as hemagglutination and neuraminidase inhibition assays. RESULTS: N-acetyl cysteine, thyme and pelargonium extract showed no or only marginal cytotoxicity in MDCK and HeLa Ohio cells in the tested concentration range. The 50% cytotoxic concentration of ambroxol and bromhexine was 51.85 and 61.24 µM, respectively. No anti-rhinoviral activity was detected at non-cytotoxic concentrations in this in vitro study setting. Ambroxol, bromhexine, and N-acetyl cysteine inhibited the influenza virus-induced cytopathic effect in MDCK cells no or less than 50%. In contrast, a dose-dependent anti-influenza virus activity of thyme and pelargonium extracts was demonstrated. The time-of addition assays revealed an inhibition of early and late steps of influenza virus replication by pelargonium extract whereas zanamivir acted on late steps only. The proven block of viral neuraminidase activity might explain the inhibition of influenza virus replication when added after viral adsorption. CONCLUSION: The study results indicate a distinct inhibition of influenza A virus replication by thyme and pelargonium extract which might contribute to the beneficial effects of these plant extracts on acute respiratory infections symptoms.

A universal dual mechanism immunotherapy for the treatment of influenza virus infections.

Seasonal influenza epidemics lead to 3-5 million severe infections and 290,000-650,000 annual global deaths. With deaths from the 1918 influenza pandemic estimated at >50,000,000 and future pandemics anticipated, the need for a potent influenza treatment is critical. In this study, we design and synthesize a bifunctional small molecule by conjugating the neuraminidase inhibitor, zanamivir, with the highly immunogenic hapten, dinitrophenyl (DNP), which specifically targets the surface of free virus and viral-infected cells. We show that this leads to simultaneous inhibition of virus release, and immune-mediated elimination of both free virus and virus-infected cells. Intranasal or intraperitoneal administration of a single dose of drug to mice infected with 100x MLD50 virus is shown to eradicate advanced infections from representative strains of both influenza A and B viruses. Since treatments of severe infections remain effective up to three days post lethal inoculation, our approach may successfully treat infections refractory to current therapies.

Type-IInterferon-Inducible SERTAD3 Inhibits Influenza A Virus Replication by Blocking the Assembly of Viral RNA Polymerase Complex.

Influenza A virus (IAV) infection stimulates a type I interferon (IFN-I) response in host cells that exerts antiviral effects by inducing the expression of hundreds of IFN-stimulated genes (ISGs). However, most ISGs are poorly studied for their roles in the infection of IAV. Herein, we demonstrate that SERTA domain containing 3 (SERTAD3) has a significant inhibitory effect on IAV replication in vitro. More importantly, Sertad3-/- mice develop more severe symptoms upon IAV infection. Mechanistically, we find SERTAD3 reduces IAV replication through interacting with viral polymerase basic protein 2 (PB2), polymerase basic protein 1 (PB1), and polymerase acidic protein (PA) to disrupt the formation of the RNA-dependent RNA polymerase (RdRp) complex. We further identify an 8-amino-acid peptide of SERTAD3 as a minimum interacting motif that can disrupt RdRp complex formation and inhibit IAV replication. Thus, our studies not only identify SERTAD3 as an antiviral ISG, but also provide the mechanism of potential application of SERTAD3-derived peptide in suppressing influenza replication.