Antiviral activity of pimecrolimus against dengue virus type 2 infection in vitro and in vivo.

Dengue virus (DENV) infection is a public health concern in several countries and is associated with severe diseases, such as dengue hemorrhagic fever and dengue shock syndrome. DENVs are transmitted to humans via the bites of infected Aedes mosquitoes, and no antiviral therapeutics are currently available. In this work, we aimed to identify antiviral drugs against DENV type 2 (DENV2) infections and selected pimecrolimus as a potential antiviral drug candidate. Pimecrolimus significantly inhibited DENV2-mediated cell death and replication in vitro. We also confirmed a decrease in the number of plaques formed as well as in the envelope protein levels of DENV2. The time-of-addition and course experiments revealed that pimecrolimus inhibited DENV2 infection during the early stages of the virus replication cycle. In an experimental mouse model, orally administered pimecrolimus alleviated body weight loss and lethality caused by DENV2 infection, which we used as readouts of the drug's antiviral potency. Furthermore, pimecrolimus significantly inhibited the DENV2 load and ameliorated focal necrosis in the liver and spleen. Taken together, our in vitro and in vivo findings suggest that pimecrolimus is a promising antiviral drug candidate for the treatment of DENV2 infection.

Antiviral Mechanisms of Saucerneol from Saururus chinensis against Enterovirus A71, Coxsackievirus A16, and Coxsackievirus B3: Role of Mitochondrial ROS and the STING/TKB-1/IRF3 Pathway.

Enterovirus A71 (EV71), coxsackievirus A16 (CVA16), and coxsackievirus B3 (CVB3) are pathogenic members of the Picornaviridae family that cause a range of diseases, including severe central nervous system complications, myocarditis, and pancreatitis. Despite the considerable public health impact of these viruses, no approved antiviral treatments are currently available. In the present study, we confirmed the potential of saucerneol, a compound derived from Saururus chinensis, as an antiviral agent against EV71, CVA16, and CVB3. In the in vivo model, saucerneol effectively suppressed CVB3 replication in the pancreas and alleviated virus-induced pancreatitis. The antiviral activity of saucerneol is associated with increased mitochondrial ROS (mROS) production. In vitro inhibition of mROS generation diminishes the antiviral efficacy of saucerneol. Moreover, saucerneol treatment enhanced the phosphorylation of STING, TBK-1, and IRF3 in EV71- and CVA16-infected cells, indicating that its antiviral effects were mediated through the STING/TBK-1/IRF3 antiviral pathway, which was activated by increased mROS production. Saucerneol is a promising natural antiviral agent against EV71, CVA16, and CVB3 and has potential against virus-induced pancreatitis and myocarditis. Further studies are required to assess its safety and efficacy, which is essential for the development of effective antiviral strategies against these viruses.

SARS-CoV-2 spike trimer vaccine expressed in Nicotiana benthamiana adjuvanted with Alum elicits protective immune responses in mice.

The ongoing coronavirus disease 2019 (COVID-19) pandemic has spurred rapid development of vaccines as part of the public health response. However, the general strategy used to construct recombinant trimeric severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike (S) proteins in mammalian cells is not completely adaptive to molecular farming. Therefore, we generated several constructs of recombinant S proteins for high expression in Nicotiana benthamiana. Intramuscular injection of N. benthamiana-expressed Sct vaccine (NSct Vac) into Balb/c mice elicited both humoral and cellular immune responses, and booster doses increased neutralizing antibody titres. In human angiotensin-converting enzyme knock-in mice, two doses of NSct Vac induced anti-S and neutralizing antibodies, which cross-neutralized Alpha, Beta, Delta and Omicron variants. Survival rates after lethal challenge with SARS-CoV-2 were up to 80%, without significant body weight loss, and viral titres in lung tissue fell rapidly, with no infectious virus detectable at 7-day post-infection. Thus, plant-derived NSct Vac could be a candidate COVID-19 vaccine.

Obesity Exacerbates Coxsackievirus Infection via Lipid-Induced Mitochondrial Reactive Oxygen Species Generation.

Coxsackievirus B3 (CVB3) infection causes acute pancreatitis and myocarditis. However, its pathophysiological mechanism is unclear. Here, we investigated how lipid metabolism is associated with exacerbation of CVB3 pathology using high-fat diet (HFD)-induced obese mice. Mice were intraperitoneally inoculated with 1×106 pfu/mouse of CVB3 after being fed a control or HFD to induce obesity. Mice were treated with mitoquinone (MitoQ) to reduce the level of mitochondrial ROS (mtROS). In obese mice, lipotoxicity of white adipose tissue-induced inflammation caused increased replication of CVB3 and mortality. The coxsackievirus adenovirus receptor increased under obese conditions, facilitating CVB3 replication in vitro. However, lipid-treated cells with receptor-specific inhibitors did not reduce CVB3 replication. In addition, lipid treatment increased mitochondria-derived vesicle formation and the number of multivesicular bodies. Alternatively, we found that inhibition of lipid-induced mtROS decreased viral replication. Notably, HFD-fed mice were more susceptible to CVB3-induced mortality in association with increased levels of CVB3 replication in adipose tissue, which was ameliorated by administration of the mtROS inhibitor, MitoQ. These results suggest that mtROS inhibitors can be used as potential treatments for CVB3 infection.

Structure and antiviral activity of a pectic polysaccharide from the root of Sanguisorba officinalis against enterovirus 71 in vitro/vivo.

The increasing prevalence and pandemic risk of viral diseases warrant the development of safe and effective treatments. In this study, we aimed to elucidate the structure and anti-enterovirus 71 (EV71) effects of polysaccharides isolated from the roots of Sanguisorba officinalis (SO), traditionally used for infectious diseases. The purified polysaccharide (S-a3) was a homogenous macromolecule (260.4 kDa) with a concave and porous surface. Linkage and NMR analyses confirmed that S-a3 is a polysaccharide interlinked with homogalacturonan, rhamnogalacturonan-I, 1,4-α-glucan, and arabinogalactan. S-a3 significantly inhibited cell death and viral gene expression in EV71-infected Vero cells, and alleviated EV71-induced body weight loss, death, and paralysis in the hSCARB2-transgenic mouse model. The effective dose of S-a3 was non-toxic to cells and mice. The antiviral mechanism of S-a3 was associated with the disruption of EV71 attachment to host cells. Our findings demonstrate that polysaccharides from SO can be a safe and effective treatment for EV71 infection.

Antiviral Activity of Chrysin against Influenza Virus Replication via Inhibition of Autophagy.

Influenza viruses cause respiratory infections in humans and animals, which have high morbidity and mortality rates. Although several drugs that inhibit viral neuraminidase are used to treat influenza infections, the emergence of resistant viruses necessitates the urgent development of new antiviral drugs. Chrysin (5,7-dihydroxyflavone) is a natural flavonoid that exhibits antiviral activity against enterovirus 71 (EV71) by inhibiting viral 3C protease activity. In this study, we evaluated the antiviral activity of chrysin against influenza A/Puerto Rico/8/34 (A/PR/8). Chrysin significantly inhibited A/PR/8-mediated cell death and the replication of A/PR/8 at concentrations up to 2 µM. Viral hemagglutinin expression was also markedly decreased by the chrysin treatment in A/PR/8-infected cells. Through the time course experiment and time-of-addition assay, we found that chrysin inhibited viral infection at the early stages of the replication cycle. Additionally, the nucleoprotein expression of A/PR/8 in A549 cells was reduced upon treatment with chrysin. Regarding the mechanism of action, we found that chrysin inhibited autophagy activation by increasing the phosphorylation of mammalian target of rapamycin (mTOR). We also confirmed a decrease in LC3B expression and LC3-positive puncta levels in A/PR/8-infected cells. These results suggest that chrysin exhibits antiviral activity by activating mTOR and inhibiting autophagy to inhibit the replication of A/PR/8 in the early stages of infection.

Morin Hydrate Inhibits Influenza Virus entry into Host Cells and Has Anti-inflammatory Effect in Influenza-infected Mice.

Influenza virus is the major cause of seasonal and pandemic flu. Currently, oseltamivir, a potent and selective inhibitor of neuraminidase of influenza A and B viruses, is the drug of choice for treating patients with influenza virus infection. However, recent emergence of oseltamivir-resistant influenza viruses has limited its efficacy. Morin hydrate (3,5,7,2',4'-pentahydroxyflavone) is a flavonoid isolated from Morus alba L. It has antioxidant, anti-inflammatory, neuroprotective, and anticancer effects partly by the inhibition of the NF-кB signaling pathway. However, its effects on influenza virus have not been studied. We evaluated the antiviral activity of morin hydrate against influenza A/Puerto Rico/8/1934 (A/PR/8; H1N1) and oseltamivir-resistant A/PR/8 influenza viruses in vitro. To determine its mode of action, we carried out time course experiments, and time of addition, hemolysis inhibition, and hemagglutination assays. The effects of the co-administration of morin hydrate and oseltamivir were assessed using the murine model of A/PR/8 infection. We found that morin hydrate reduced hemagglutination by A/PR/8 in vitro. It alleviated the symptoms of A/PR/8-infection, and reduced the levels of pro-inflammatory cytokines and chemokines, such as TNF-α and CCL2, in infected mice. Co-administration of morin hydrate and oseltamivir phosphate reduced the virus titers and attenuated pulmonary inflammation. Our results suggest that morin hydrate exhibits antiviral activity by inhibiting the entry of the virus.

Photoreaction characteristics of ferrous oxalate recovered from wastewater.

Photoreaction with the Fe(III)-oxalate complex has been reported to play an important role in various photochemical reactions in the natural atmospheric environment, and are applicable to treat various recalcitrant compounds in wastewater. We previously showed that a Fe(II) oxalate (FeOx) crystal can be recovered from the wastewater generated from soil washing, which can then be applied to the photoreaction for degradation of recalcitrant pollutants; however, photoreactions with FeOx compounds have not been fully reported yet. Therefore, this study aims to investigate the photoreaction characteristics of FeOx recovered from wastewater, to demonstrate the feasibility of its application to wastewater treatment. The physical and chemical properties of FeOx were characterized with X-ray diffraction, scanning electron microscopy/energy-dispersive X-ray spectrometry, and X-ray photoelectron spectroscopy analyses. The photoreaction of FeOx showed high methylene blue (MB) removal efficiencies similar to the reaction with TiO2, indicating that FeOx is applicable to the photoreaction for degradation of pollutants. Furthermore, the photodegradation of MB with FeOx was observed under visible light, as opposed to TiO2. MB could be removed by the photoreaction of FeOx under both basic and acidic pH conditions. Under basic pH conditions, MB could be removed by FeOx via both photoreaction and surface adsorption. The concentration of FeOx affected light penetration and Fe and oxalate levels in the solution, resulting in different MB removal kinetics. The photoreaction efficiency of FeOx could be affected by both photoreaction of Fe and oxalate in the solution and photoreaction and adsorption reaction of the FeOx surface.

Local Stabilization of Hypoxia-Inducible Factor-1α Controls Intestinal Inflammation via Enhanced Gut Barrier Function and Immune Regulation.

Intestinal epithelial cells are adapted in mucosal hypoxia and hypoxia-inducible factors in these cells can fortify barrier integrity to support mucosal tissue healing. Here we investigated whether hypoxia-related pathways could be proposed as potential therapeutic targets for inflammatory bowel disease. We developed a novel hypoxia-inducible factor (HIF) prolyl hydroxylase inhibitor, CG-598 which stabilized HIF-1α in the gut tissue. Treatment of CG-598 did not affect extra-intestinal organs or cause any significant adverse effects such as erythropoiesis. In the experimental murine colitis model, CG-598 ameliorated intestinal inflammation with reduction of inflammatory lesions and pro-inflammatory cytokines. CG-598 treatment fortified barrier function by increasing the expression of intestinal trefoil factor, CD73, E-cadherin and mucin. Also, IL-10 and IL-22 were induced from lamina propria CD4+ T-cells. The effectiveness of CG-598 was comparable to other immunosuppressive therapeutics such as TNF-blockers or JAK inhibitors. These results suggest that CG-598 could be a promising therapeutic candidate to treat inflammatory bowel disease.

ERdj5 in Innate Immune Cells Is a Crucial Factor for the Mucosal Adjuvanticity of Cholera Toxin.

Cholera toxin (CT) is one of most strong mucosal adjuvants, but it cannot be clinically used owing to its toxicity. The cytosolic A1 subunit of CT (CTA1) is the molecule responsible for its immunostimulatory activity, which increases the concentration of cyclic AMP and causes the induction of pro-inflammatory cytokines in innate immune cells. However, the importance of endoplasmic reticulum (ER) molecules involved in CTA1 retro-translocation to induce immune responses remained to be investigated. ERdj5 is an ER protein which is expected to transfer CTA1 to the Hrd1 complex for the retro-translocation of CTA1. In this study, we investigated the physiological relevance of ERdj5 in immune stimulation by CT. ERdj5-knockout (ERdj5 KO) mice had decreased production of antigen-specific IgG in the serum and IgA in the mucosal secretion after intranasal immunization with Ag and CT. Especially, IgG2c isotypes were specifically reduced in the absence of ERdj5. ERdj5 KO dendritic cells (DCs) failed to full activation with decreased expression of costimulatory molecules, such as MHC class II, CD80, and CD 86. In ERdj5 KO DCs, secretion of pro-inflammatory cytokines, such as IL-1ß, TNF-α, and IL-6, was reduced. The cytokine signatures of several helper T cells were reduced in ERdj5 KO mice following intranasal CT immunization. The absence of ERdj5 affects the immunostimulatory properties of CT but does not affect the response to the CTB pentamer, the response to alum, total antibody production, or cytokine release from DCs exposed to CpG. Interestingly, CT enhanced the expression of ER stress proteins in ERdj5 KO innate immune cells. These results suggested that ERdj5 contributed as a decisive factor to the immunostimulatory capacity of CT via CTA1 retro-translocation.

Manassantin B shows antiviral activity against coxsackievirus B3 infection by activation of the STING/TBK-1/IRF3 signalling pathway.

Coxsackievirus B3 (CVB3) is an important human pathogen associated with the development of acute pancreatitis, myocarditis, and type 1 diabetes. Currently, no vaccines or antiviral therapeutics are approved for the prevention and treatment of CVB3 infection. We found that Saururus chinensis Baill extract showed critical antiviral activity against CVB3 infection in vitro. Further, manassantin B inhibited replication of CVB3 and suppressed CVB3 VP1 protein expression in vitro. Additionally, oral administration of manassantin B in mice attenuated CVB3 infection-associated symptoms by reducing systemic production of inflammatory cytokines and chemokines including TNF-α, IL-6, IFN-γ, CCL2, and CXCL-1. We found that the antiviral activity of manassantin B is associated with increased levels of mitochondrial ROS (mROS). Inhibition of mROS generation attenuated the antiviral activity of manassantin B in vitro. Interestingly, we found that manassantin B also induced cytosolic release of mitochondrial DNA based on cytochrome C oxidase DNA levels. We further confirmed that STING and IRF-3 expression and STING and TBK-1 phosphorylation were increased by manassantin B treatment in CVB3-infected cells. Collectively, these results suggest that manassantin B exerts antiviral activity against CVB3 through activation of the STING/TKB-1/IRF3 antiviral pathway and increased production of mROS.

Lignan Dimers from Forsythia viridissima Roots and Their Antiviral Effects.

Six new dimeric lignans (1-6) and one new lignan glycoside (16) were isolated from Forsythia viridissima roots along with nine known lignans (7-15). Spectroscopic analyses and chemical methods were used to determine these new structures and their absolute configurations. Among these compounds, dimatairesinol (1) and viridissimaols A-E (2-6) were assigned as dimers of dibenzylbutyrolactone analogues. Furthermore, the isolated compounds were evaluated for their antiviral activities against coxsackievirus B3 (CVB3) and human rhinovirus 1B (HRV1B). In these tests, compounds 12 and 15 showed antiviral effects against CVB3 infection with IC50 values of 15.4 and 36.4 µM, respectively, while 2, 3, 8, and 9 showed activities against HRV1B with IC50 values of 45.7, 47.5, 13.0, and 43.2 µM, respectively.

Anti-Human Rhinovirus 1B Activity of Dexamethasone viaGCR-Dependent Autophagy Activation.

OBJECTIVES: Human rhinoviruses (HRVs) are the major cause of the common cold. Currently there is no registered, clinically effective, antiviral chemotherapeutic agent to treat diseases caused by HRVs. In this study, the antiviral activity of dexamethasone (DEX) against HRV1B was examined. METHODS: The anti-HRV1B activity of DEX was assessed by sulforhodamine B assay in HeLa cells, and by RT-PCR in the lungs of HRV1B-infected mice. Histological evaluation of HRV1B-infected lungs was performed and a histological score was given. Anti-HRV1B activity of DEX via the glucocorticoid receptor (GCR)-dependent autophagy activation was assessed by blocking with chloroquine diphosphate salt or bafilomycin A1 treatment. RESULTS: In HRV1B-infected HeLa cells, treatment with DEX in a dose-dependent manner, resulted in a cell viability of > 70% indicating that HRV1B viral replication was reduced by DEX treatment. HRV1B infected mice treated with DEX, had evidence of reduced inflammation and a moderate histological score. DEX treatment showed antiviral activity against HRV1B via GCR-dependent autophagy activation. CONCLUSION: This study demonstrated that DEX treatment showed anti-HRV1B activity via GCR-dependent autophagy activation in HeLa cells and HRV1B infected mice. Further investigation assessing the development of topical formulations may enable the development of improved DEX effectiveness.

Antiviral and anti-inflammatory activity of budesonide against human rhinovirus infection mediated via autophagy activation.

Human rhinovirus (HRV) infection causes more than 80% of all common colds and is associated with severe complications in patients with asthma and chronic obstructive pulmonary disease. To identify antiviral drug against HRV infection, we screened 800 FDA-approved drugs and found budesonide as one of the possible drug candidates. Budesonide is a corticosteroid, which is commonly used to prevent exacerbation of asthma and symptoms of common cold. Budesonide specifically protects host cells from cytotoxicity following HRV infection, which depend on the expression of glucocorticoid receptor. Intranasal administration of budesonide lowered the pulmonary HRV load and the levels of IL-1ß cytokine leading to decreased lung inflammation. Budesonide regulates IL-1ß production following HRV infection independent of inflammasome activation. Instead, budesonide induces mitochondrial reactive oxygen species followed by activation of autophagy. Further, the inhibition of autophagy following chloroquine or bafilomycin A1 treatment reduced the anti-viral effect of budesonide against HRV, suggesting that the antiviral activity of budesonide was mediated via autophagy. The results suggest that budesonide represents a promising antiviral and anti-inflammatory drug candidate for the treatment of human rhinovirus infection.

Antiviral activity of gemcitabine against human rhinovirus in vitro and in vivo.

Rhinovirus, a major causative agent of the common cold, is associated with exacerbation of asthma and chronic obstructive pulmonary disease. Currently, there is no antiviral treatment or vaccine for human rhinovirus (HRV). Gemcitabine (2',2'-difluorodeoxycytidine, dFdC) is a deoxycytidine analog with antiviral activity against rhinovirus, as well as enterovirus 71, in vitro. However, the antiviral effects of gemcitabine in vivo have not been investigated. In the current study, we assessed whether gemcitabine mediated antiviral effects in the murine HRV infection model. Intranasal administration of gemcitabine significantly lowered pulmonary viral load and inflammation by decreasing proinflammatory cytokines, including TNF-α and IL-1ß, and reduction in the number of lung-infiltrating lymphocytes. Interestingly, we found that the addition of UTP and CTP significantly attenuated the antiviral activity of gemcitabine. Thus the limitation of UTP and CTP by the addition of gemcitabine may inhibit the viral RNA synthesis. These results suggest that gemcitabine, an antineoplastic drug, can be repositioned as an antiviral drug to inhibit HRV infection.

Ready-to-use colloidal adjuvant systems for intranasal immunization.

Adjuvant systems based on oil-in-water (o/w) microemulsions (MEs) for vaccination via intranasal administration were prepared and evaluated. A ready-to-use blank ME system composed of mineral oil (oil), Labrasol (surfactant), Tween 80 (cosurfactant), and water was prepared and blended with antigen (Ag) solution prior to use. The o/w ME system developed exhibited nano-size droplets within the tested range of Ag concentrations and dilution factors. The maintenance of primary, secondary, and tertiary structural stability of ovalbumin (OVA) in ME, compared with OVA in solution, was demonstrated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), circular dichroism (CD), and fluorescence intensity measurements, respectively. The uptake efficiency in RAW 264.7 cells, evaluated by flow cytometry, of OVA in the ME group was significantly higher than that of the OVA solution group (p<0.05). In an intranasal immunization study with OVA ME in mice, elevated adjuvant effects in terms of mucosal immunization and Th1-dominant cell-mediated immune responses were identified. Given the convenience of use (simply mixing with Ag solution prior to use) and the adjuvant effects after intranasal immunization, the new o/w ME may be a practical and efficient adjuvant system for intranasal vaccination.

Complete mitochondrial genome of the nerippe fritillary butterfly, Argynnis nerippe (Lepidoptera: Nymphalidae).

The complete mitochondrial genome sequence of the nerippe fritillary butterfly, Argynnis nerippe, which is listed as an endangered species in Korea, is described with an emphasis on the A+T-rich region. The 15,140-bp long circular molecule consisted of 13 protein-coding genes, two rRNA genes, 22 tRNA genes and 1 control region, known in insect as the A+T-rich region, as found in typical metazoans. The 329-bp long A+T-rich region located between srRNA and tRNA(Met) possessed the highest A/T content (95.7%) than any other region of the genome. Along with the several conserved sequences found typically in the lepidopteran insects the genome contained one tRNA(Met)-like and tRNA(Leu)(UUR)-like sequence in the A+T-rich region.

The complete mitogenome sequence of the Japanese oak silkmoth, Antheraea yamamai (Lepidoptera: Saturniidae).

The 15,338-bp long complete mitochondrial genome (mitogenome) of the Japanese oak silkmoth, Antheraea yamamai (Lepidoptera: Saturniidae) was determined. This genome has a gene arrangement identical to those of all other sequenced lepidopteran insects, but differs from the most common type, as the result of the movement of tRNA(Met) to a position 5'-upstream of tRNA(Ile). No typical start codon of the A. yamamai COI gene is available. Instead, a tetranucleotide, TTAG, which is found at the beginning context of all sequenced lepidopteran insects was tentatively designated as the start codon for A. yamamai COI gene. Three of the 13 protein-coding genes (PCGs) harbor the incomplete termination codon, T or TA. All tRNAs formed stable stem-and-loop structures, with the exception of tRNA(Ser)(AGN), the DHU arm of which formed a simple loop as has been observed in many other metazoan mt tRNA(Ser)(AGN). The 334-bp long A + T-rich region is noteworthy in that it harbors tRNA-like structures, as has also been seen in the A + T-rich regions of other insect mitogenomes. Phylogenetic analyses of the available species of Bombycoidea, Pyraloidea, and Tortricidea bolstered the current morphology-based hypothesis that Bombycoidea and Pyraloidea are monophyletic (Obtectomera). As has been previously suggested, Bombycidae (Bombyx mori and B. mandarina) and Saturniidae (A. yamamai and Caligula boisduvalii) formed a reciprocal monophyletic group.