Anti-influenza Effect Research Articles

Anti-inflammatory effects of rosmarinic acid-4-O-β-D-glucoside in reducing acute lung injury in mice infected with influenza virus

Rosmarinic acid-4-O-β-D-glucoside (RAG) is a dicaffeoyl phenolic compound isolated from Sarcandra glabra (Thunb.) Nakai. Preliminary studies show that RAG has significant anti-inflammatory properties and can alleviate ear swelling in mice and the paw swelling in rats. Here, the anti-influenza effects of RAG were investigated in mice infected with A/FM/1/47 H1N1 virus. The survival rate and body weight were observed, the lung edema, virus copies, inflammatory cytokines (including IL-4, IL-5, TNF-α and IFN-γ) and oxidative damage indexes (including SOD, MDA, NO, and CAT) were measured. Moreover, immune cell recruitment in alveoli was measured with white blood cells and differential counts. Therapeutic RAG concentrations substantially improve the symptoms, mitigate body weight loss and alleviate lung edema induced by virus, thus improve survival protection effects. Furthermore, RAG was shown to regulate influenza virus-induced inflammatory cytokine expression, specifically by downregulating the Th1 cell cytokines IFN-γ, TNF-α and upregulating the Th2 cell cytokines IL-4, IL-5. Cell migration and infiltration were also diminished after RAG administration.

Essential Oil Composition and Bioactivities of Waldheimia glabra (Asteraceae) from Qinghai-Tibet Plateau.

Waldheimia glabra is traditionally used as incense and as an anti-influenza drug by Tibetans in China. Here, we collected W. glabra from the Gangs Rinpoche mountain at an altitude of 5200 m, and analyzed its essential oil by gas chromatography-mass spectrometry (GC-MS) combined with the retention indices (RI). Twenty-seven compounds, representing 72.4% of the total essential oil, were identified, including α-bisabolol (20.2%), valeranone (11.8%), chamazulene (9.9%), spathulenol (8.2%), β-caryophyllene (6.1%), and caryophyllene oxide (5.2%). Bioactivity evaluation of the essential oil revealed that it exhibited potent anti-influenza effect on viruses H3N2 and anti-inflammatory effect by inhibiting the lipopolysaccharide (LPS)-induced nitric oxide (NO) production in RAW 264.7 macrophages, but no anti-complementary activity.

Identification of small molecule inhibitors for influenza a virus using in silico and in vitro approaches.

Influenza viruses have acquired resistance to approved neuraminidase-targeting drugs, increasing the need for new drug targets for the development of novel anti-influenza drugs. Nucleoprotein (NP) is an attractive target since it has an indispensable role in virus replication and its amino acid sequence is well conserved. In this study, we aimed to identify new inhibitors of the NP using a structure-based drug discovery algorithm, named Nagasaki University Docking Engine (NUDE), which has been established especially for the Destination for GPU Intensive Machine (DEGIMA) supercomputer. The hit compounds that showed high binding scores during in silico screening were subsequently evaluated for anti-influenza virus effects using a cell-based assay. A 4-hydroxyquinolinone compound, designated as NUD-1, was found to inhibit the replication of influenza virus in cultured cells. Analysis of binding between NUD-1 and NP using surface plasmon resonance assay and fragment molecular orbital calculations confirmed that NUD-1 binds to NP and could interfere with NP-NP interactions essential for virus replication. Time-of-addition experiments showed that the compound inhibited the mid-stage of infection, corresponding to assembly of the NP and other viral proteins. Moreover, NUD-1 was also effective against various types of influenza A viruses including a clinical isolate of A(H1N1)pdm09 influenza with a 50% inhibitory concentration range of 1.8–2.1 μM. Our data demonstrate that the combined use of NUDE system followed by the cell-based assay is useful to obtain lead compounds for the development of novel anti-influenza drugs.

In vitro Anti-Influenza Virus Activity of Agaricus brasiliensis KA21.

Agaricus is known to have immunostimulatory and anti-tumor effects. However, the antiviral effects of Agaricus have not yet been examined. In the present study, the antiviral effects of an extract of Agaricus brasiliensis KA21 (AE) on the H1N1 influenza virus (PR8 strain) were investigated. The anti-influenza virus effects of AE were examined by using the plaque formation inhibition test. AE inhibited the plaque formation of PR8 in a dose-dependent manner: 98 and 50% (IC50) inhibition at 2.5 and 0.99 mg/mL, respectively. To elucidate the mechanisms of AE, the direct actions and adsorption and invasion inhibition of AE were examined, and were found to have no inhibitory effect on PR8 infection. Thus, in vitro antiviral effects may somehow inhibit PR8 after the viral invasion of cells. These results demonstrated that it is expected that AE can effectively prevent the spread of the influenza virus.

Anti-flu Effect of Compound Yizhihao Granule and Its Effective Components

ObjectiveTo research the anti-influenza effect of active ingredients in Compound Yizhihao Granule (CYG). MethodsThe cytotoxicity and cytopathic effects (CPE) were observed under the phase-contrast microscope, besides 50% toxicity concentration (TC50) and 50% inhibitory concentration (IC50) were also calculated using Reed-Muench method, then the antiviral activity in vitro according to Selection Index (SI = TC50/IC50) was evaluated. In PR8 virus-infected mice, survival time, death rate, and lung index were observed in order to evaluate the protective effect. Besides, the effective ingredients were determined using HPLC method, and their contents were calculated by external standard method. ResultsCYG could inhibit the influenza virus-induced CPE, with IC50 of 4.6 mg/mL (equal to herbal extracts 262.2 μg/mL), and no direct cytotoxic effect at this concentration. PR8-infected mice were ig given CYG, the lung index and mortality were significantly reduced, and survival time was obviously prolonged. HPLC analysis indicated CYG contained many kinds of antivirus active components, including rupestonic acid, epigoitrin, and adenosine. ConclusionCYG is an effective natural anti-influenza medicine. Its antiviral effect should be the synergic effect of a variety of antiviral active ingredients.

Anti-influenza Virus Effects of Catechins: A Molecular and Clinical Review.

Influenza infection and associated epidemics represent a serious public health problem. Several preventive and curative measures exist against its spread including vaccination and therapeutic agents such as neuraminidase inhibitors (e.g., oseltamivir, zanamivir, as well as peramivir and laninamivir, which are licensed in several countries) and adamantanes (e.g., amantadine and rimantadine). However, neuraminidase inhibitor- and adamantane- resistant viruses have been detected, whereas vaccines exhibit strain-specific effects and are limited in supply. Thus, new approaches are needed to prevent and treat influenza infections. Catechins, a class of polyphenolic flavonoids present in tea leaves, have been reported as potential anti-influenza virus agents based on experimental and clinical studies. (-)-epigallocatechin gallate (EGCG), a major and highly bioactive catechin, is known to inhibit influenza A and B virus infections in Madin-Darby canine kidney cells. Additionally, EGCG and other catechin compounds such as epicatechin gallate and catechin-5-gallate also show neuraminidase inhibitory activities as demonstrated via molecular docking. These catechins can bind differently to neuraminidase and might overcome known drug resistancerelated virus mutations. Furthermore, the antiviral effects of chemically modified catechin derivatives have also been investigated, and future structure-based drug design studies of catechin derivatives might contribute to improvements in influenza prevention and treatment. This review briefly summarizes probable mechanisms underlying the inhibitory effects of tea catechins against influenza infection and their clinical benefits on influenza prevention and treatment. Additionally, the great potential of tea catechins and their chemical derivatives as effective antiviral agents is described.

Melissa officinalis efficacy against human influenza virus (New H1N1) in comparison with oseltamivir

ObjectiveTo evaluate the antiviral activity of Melissa officinalis (MO) extract against the influenza virus H1N1 in vitro. MethodsThe cytotoxicity of MO extract was identified on Madin-Darby canine kidney (MDCK) cell culture by MTT assay. The virus was inoculated to the cells (multiplicity of infection = 0.1) in two protocols. In protocol 1, the MO extracts at concentrations of 0.005, 0.050, 0.010, 0.100 and 0.500 mg/mL were incubated with the virus for one hour pre-inoculation. In protocol 2, the mentioned concentrations of MO extracts were added to the cells one-hour post infection. Furthermore, the antiviral effect of oseltamivir with different concentrations was tested as the positive controls. The 50% tissue culture infective dose, neutralizing index and hemagglutination titer were determined. ResultsThe medicine oseltamivir and MO extracts were not toxic for MDCK at concentrations less than 1 mg/mL. All utilized concentrations of MO extracts were vigorously efficient to decrease the viral yield in both experiments. The 50% tissue culture infective dose of the groups containing up to 0.100 mg/mL of MO extracts in the first experiment in compare with 0.050 mg/mL in the second experiment reduced to 0. Although hemagglutination tests showed little titers, the viral quantity significantly decreased in both experiments. By the way, the medicine oseltamivir could completely suppress viral replication in MDCK. ConclusionsThe present study suggests that MO extracts have a potent anti-influenza effect in cell culture.

Screening for Neuraminidase Inhibitory Activity in Traditional Chinese Medicines Used to Treat Influenza.

Objective: To screen for influenza virus neuraminidase inhibition and to provide a reference for the clinical treatment of influenza using traditional Chinese medicines (TCM). In this study, 421 crude extracts (solubilized with petroleum ether, ethanol, ethyl acetate, and aqueous solvents) were obtained from 113 TCM. The medicine extracts were then reacted with oseltamivir, using 2’-(4-methylumbelliferyl)-α-D-N-acetylneuraminic acid (MUNANA) as the substrate, to determine influenza virus neuraminidase activity using a standard fluorimetric assay. It was found that Chinese medicine extracts from Pyrola calliantha, Cynanchum wilfordii, Balanophora involucrata and Paeonia delavayi significantly inhibited neuraminidase activity at a concentration of 40 μg/mL. Dose-dependent inhibitory assays also revealed significant inhibition. The IC50 range of the TCM extracts for influenza virus neuraminidase was approximately 12.66–34.85 μg/mL, respectively. Some Chinese medicines have clear anti-influenza viral effects that may play an important role in the treatment of influenza through the inhibition of viral neuraminidase. The results of this study demonstrated that plant medicines can serve as a useful source of neuraminidase (NA) inhibitors and further investigation into the pharmacologic activities of these extracts is warranted.

Nasal commensal Staphylococcus epidermidis counteracts influenza virus.

Several microbes, including Staphylococcus epidermidis (S. epidermidis), a Gram-positive bacterium, live inside the human nasal cavity as commensals. The role of these nasal commensals in host innate immunity is largely unknown, although bacterial interference in the nasal microbiome may promote ecological competition between commensal bacteria and pathogenic species. We demonstrate here that S. epidermidis culture supernatants significantly suppressed the infectivity of various influenza viruses. Using high-performance liquid chromatography together with mass spectrometry, we identified a giant extracellular matrix-binding protein (Embp) as the major component involved in the anti-influenza effect of S. epidermidis. This anti-influenza activity was abrogated when Embp was mutated, confirming that Embp is essential for S. epidermidis activity against viral infection. We also showed that both S. epidermidis bacterial particles and Embp can directly bind to influenza virus. Furthermore, the injection of a recombinant Embp fragment containing a fibronectin-binding domain into embryonated eggs increased the survival rate of virus-infected chicken embryos. For an in vivo challenge study, prior Embp intranasal inoculation in chickens suppressed the viral titres and induced the expression of antiviral cytokines in the nasal tissues. These results suggest that S. epidermidis in the nasal cavity may serve as a defence mechanism against influenza virus infection.

A novel small-molecule compound disrupts influenza A virus PB2 cap-binding and inhibits viral replication.

The conserved residues 318-483 in the PB2 subunit of influenza A polymerase is an independently folded cap-binding domain (PB2cap) that exhibits a distinct binding mode from other host cap-binding proteins, which suggests that PB2cap might be an ideal drug target. This study aimed to identify a new class of anti-influenza inhibitors that specifically disrupts the interaction between PB2cap and host cap structures. An innovative fluorescence polarization assay was established for primary screening, followed by cap-binding inhibitory activity, antiviral efficacy and cytotoxicity evaluations of the selected compounds. The best compound was characterized by multi-cycle virus growth assay, cross-protection test, synergism evaluation, mini-replicon assay, binding affinity analysis, docking simulation and mouse study. Several PB2 cap-binding inhibitors were discovered. The compound 7-(4-hydroxy-2-oxo-2H-chromen-3-yl)-6H,7H,8H-chromeno[3',4':5,6]pyrano[3,2-c]chromene-6,8-dione, designated PB2-39, was identified as a potent inhibitor of replication of multiple subtypes of influenza A virus, including H1N1, H3N2, H5N1, H7N7, H7N9 and H9N2 in vitro and H1N1, H5N1 and H7N9 in vivo. Combinational treatment with the influenza virus release inhibitor zanamivir and PB2-39 exerted a synergistic anti-influenza effect. Mechanistic experiments supported that PB2-39 suppressed viral polymerase activity. Docking and binding affinity analyses demonstrated that PB2-39 interacted with the PB2 cap-binding pocket, suggesting its role as a cap-binding competitor. Our study provides new insights for the strategic development of novel cap-binding inhibitors of influenza A viruses.

Investigating the role of MRGPRC11 and capsaicin-sensitive afferent nerves in the anti-influenza effects exerted by SLIGRL-amide in murine airways.

BackgroundThe hexapeptide SLIGRL-amide activates protease-activated receptor-2 (PAR-2) and mas-related G protein-coupled receptor C11 (MRGPRC11), both of which are known to be expressed on populations of sensory nerves. SLIGRL-amide has recently been reported to inhibit influenza A (IAV) infection in mice independently of PAR-2 activation, however the explicit roles of MRGPRC11 and sensory nerves in this process are unknown. Thus, the principal aim of this study was to determine whether SLIGRL-amide-induced inhibition of influenza infection is mediated by MRGPRC11 and/or by capsaicin-sensitive sensory nerves.MethodsThe inhibitory effect of SLIGRL-amide on IAV infection observed in control mice in vivo was compared to effects produced in mice that did not express MRGPRC11 (mrgpr-cluster∆−/− mice) or had impaired sensory nerve function (induced by chronic pre-treatment with capsaicin). Complementary mechanistic studies using both in vivo and ex vivo approaches investigated whether the anti-IAV activity of SLIGRL-amide was (1) mimicked by either activators of MRGPRC11 (BAM8-22) or by activators (acute capsaicin) or selected mediators (substance P, CGRP) of sensory nerve function, or (2) suppressed by inhibitors of sensory nerve function (e.g. NK1 receptor antagonists).ResultsSLIGRL-amide and BAM8-22 dose-dependently inhibited IAV infection in mrgpr-cluster∆−/− mice that do not express MRGPRC11. In addition, SLIGRL-amide and BAM8-22 each inhibited IAV infection in capsaicin-pre-treated mice that lack functional sensory nerves. Furthermore, the anti-IAV activity of SLIGRL-amide was not mimicked by the sensory neuropeptides substance P or CGRP, nor blocked by either NK1 (L-703,606, RP67580) and CGRP receptor (CGRP8-37) antagonists. Direct stimulation of airway sensory nerves through acute exposure to the TRPV1 activator capsaicin also failed to mimic SLIGRL-amide-induced inhibition of IAV infectivity. The anti-IAV activity of SLIGRL-amide was mimicked by the purinoceptor agonist ATP, a direct activator of mucus secretion from airway epithelial cells. Additionally, both SLIGRL-amide and ATP stimulated mucus secretion and inhibited IAV infectivity in mouse isolated tracheal segments.ConclusionsSLIGRL-amide inhibits IAV infection independently of MRGPRC11 and independently of capsaicin-sensitive, neuropeptide-releasing sensory nerves, and its secretory action on epithelial cells warrants further investigation.

Host-directed therapies for antimicrobial resistant respiratory tract infections.

Antimicrobial-resistant respiratory tract infections (AMR-RTIs) are increasing, presenting important management challenges worldwide. Current management of AMR-RTI patients focuses on pathogen-directed antimicrobial treatment. Overt lung inflammation, parenchymal damage, and ineffective immune activation perpetrate increased patient morbidity and mortality. Immunomodulatory and tissue-regenerative host-directed therapies (HDT) may improve treatment outcomes. HDTs under investigation for improving AMR-RTI treatment outcomes are reviewed. Various HDTs are being developed or evaluated for adjunctive AMR-RTI treatment. α-1 antitrypsin was shown to reduce Pseudomonas aeruginosa burden in the airways of cystic fibrosis patients. Cellular therapy by reinfusing autologous bone marrow-derived MSCs into MDR/XDR-TB patients shows promise, whereas adjunctive T cell-based therapies are considered. Cytotoxic therapy using etoposide, a topoisomerase II-inhibiting anticancer drug extends survival of patients with severe influenza H1N1 infection-induced hemophagocytic lymphohistiocytosis. Two other novel HDT candidates, DAS181 and resveratrol show antiinfluenza effects. Novel kinase inhibitors SB203580 (MAPK-2 antagonist) and LY294002 (phosphoinositide-3 kinases antagonist) exhibit promising anti-MERS-CoV activity. Palivizumab, an anti-RSV monoclonal antibody, effectively prevents RSV infection in high-risk paediatric populations. T-cell therapy is currently considered for adjunctive HDT of azole-resistant pulmonary aspergillosis. Novel HDTs may revolutionize future treatment regimens for AMR-RTIs. Well designed multisite clinical trials are now necessary to accelerate progress.

A novel small-molecule inhibitor of influenza A virus acts by suppressing PA endonuclease activity of the viral polymerase.

The RNA-dependent RNA polymerase of influenza A virus comprises conserved and independently-folded subdomains with defined functionalities. The N-terminal domain of the PA subunit (PAN) harbors the endonuclease function so that it can serve as a desired target for drug discovery. To identify a class of anti-influenza inhibitors that impedes PAN endonuclease activity, a screening approach that integrated the fluorescence resonance energy transfer based endonuclease inhibitory assay with the DNA gel-based endonuclease inhibitory assay was conducted, followed by the evaluation of antiviral efficacies and potential cytotoxicity of the primary hits in vitro and in vivo. A small-molecule compound ANA-0 was identified as a potent inhibitor against the replication of multiple subtypes of influenza A virus, including H1N1, H3N2, H5N1, H7N7, H7N9 and H9N2, in cell cultures. Combinational treatment of zanamivir and ANA-0 exerted synergistic anti-influenza effect in vitro. Intranasal administration of ANA-0 protected mice from lethal challenge and reduced lung viral loads in H1N1 virus infected BALB/c mice. In summary, ANA-0 shows potential to be developed to novel anti-influenza agents.

Anti-H1N1 influenza effects and its possible mechanism of Huanglian Xiangru Decoction

Ethnopharmacological relevanceHuangliang Xiangru Decoction (HXD), which is described in a famous TCM monograph “Book of Nanyang for Life Savin”, is frequently used for treating cold in summer and summer heat-dampness. Aim of the studyTo date, no pharmacological study on the anti-H1N1 influenza properties of HXD has been reported. The aim of the present study was to evaluate the therapeutic action of HXD on HIN1-induced acute pulmonary inflammation and its anti-influenza mechanism focus to TLRs signal pathway in a mouse model. Materials and methodsthe mice were intranasally infected with influenza virus to induce viral pneumonia, and then treated with different doses of HXD. The Lung index and pathological changes in the lung tissue of mice were investigated to value the anti-influenza virus effect of HXD. The concentrations of cytokines (IL-2, IL-6, TNF-α and IFN-γ) and anti-oxidant factor (NO, SOD and GSH) in serum of mice were determined with ELISA. RT-PCR and Western blot were used to determine the mRNA and protein expression of TLR3, TLR7, MyD88,TRAF3 and NF-κB p65 in the lung tissues, which are the key targets of TLRs pathway. ResultsCompared with the infection group, the lung index of mice in ribavirin group, HXD high dose group and HXD middle dose group were significantly decreased, the lung indexes of these groups were 10.36±1.14mg/g, 9.89±0.79mg/g, and 10.97±0.67mg/g. Moreover, pathological changes were remarkable alleviated. HXD can reduce the contents of IL-6, TNF-α and IFN-γ, NO, and increase the contents of IL-2, SOD, GSH in serum of infected-mice significantly. At the same time, HXD can reduce the mRNA and protein expression of TLR3, TLR7, MyD88,TRAF3 and NF-κB p65 in the lung tissues of infected-mice significantly. ConclusionsHXD has significant effects on H1N1 influenza by a quantity-effect relationship, and plays its anti-influenza effect by enhancing the body’s antioxidant capacity, regulating the body's immune function and the host's TLRs pathway.

Toll-like receptor 9 ligand D-type oligodeoxynucleotide D35 as a broad inhibitor for influenza A virus replication that is associated with suppression of neuraminidase activity

The most effective drugs available to treat influenza are neuraminidase (NA) inhibitors, which provide important additional measures for the control of influenza virus infections. However, since the emergence of NA inhibitor-resistant viruses may compromise the clinical utility of this class of anti-influenza agents, it is very important to develop new anti-influenza agents which target a different region in NA responsible for its sensitivity from that for NA inhibitors and could be used to treat NA inhibitors-resistant isolates. The oligodeoxynucleotide D35, multimerized and aggregated, suppressed replication of influenza A viruses except A/WSN/33 (WSN). The suppressive viral replication by D35 depended on G-terad and multimer formation. The range of the suppressive viral replication at the late stage, including virus assembly and release from infected cells, was much larger than that at the initial stage, viral attachment and entry. D35 suppressed NA activity of influenza A viruses. Furthermore, replacing the NA gene of A/Puerto Rico/8/34 (PR8), in which viral replication was inhibited by D35 at the late stage, with the NA gene from WSN, in which viral replication was not inhibited, eliminated the D35-dependent suppression. D35 showed an additive anti-influenza effect with oseltamivir. It was also effective in vivo. These results suggest that the influenza virus NA mainly contributse to the D35-suppressible virus release from infected cells at the late stage. In addition, because administration of D35 into the virus-infected mice suppressed viral replication and weight loss, clinical application of D35 could be considered.

Human nasal epithelial cells derived from multiple subjects exhibit differential responses to H3N2 influenza virus infection in vitro

Human nasal epithelial cells derived from multiple subjects exhibit differential responses to H3N2 influenza virus infection in vitro

Computational screen and experimental validation of anti-influenza effects of quercetin and chlorogenic acid from traditional Chinese medicine.

The Influenza A virus is a great threat for human health, while various subtypes of the virus made it difficult to develop drugs. With the development of state-of-art computational chemistry, computational molecular docking could serve as a virtual screen of potential leading compound. In this study, we performed molecular docking for influenza A H1N1 (A/PR/8/34) with small molecules such as quercetin and chlorogenic acid, which were derived from traditional Chinese medicine. The results showed that these small molecules have strong binding abilities with neuraminidase from H1N1 (A/PR/8/34). Further details showed that the structural features of the molecules might be helpful for further drug design and development. The experiments in vitro, in vivo have validated the anti-influenza effect of quercetin and chlorogenic acid, which indicating comparable protection effects as zanamivir. Taken together, it was proposed that chlorogenic acid and quercetin could be employed as the effective lead compounds for anti-influenza A H1N1.

Identification of novel membrane-associated prostaglandin E synthase-1 (mPGES-1) inhibitors with anti-influenza activities in vitro

Influenza A virus (IAV) is a major public health concern that leads to high morbidity and mortality worldwide. Despite various vaccination programs and development of drugs targeting essential viral proteins, the emergence of drug-resistant variants has been frequently reported and the therapeutic options are limited. Because exaggerated inflammation is considered as an important factor in disease pathogenesis, immunomodulatory agents that effectively suppress cytokine responses are needed for the treatment of IAV infection. Membrane-associated prostaglandin E synthase-1 (mPGES-1) is an enzyme responsible for the production of prostaglandin E2 (PGE2) that is the best-characterized immune modulatory lipid in vitro and in vivo models of inflammation. In the present study, we tested the anti-influenza activities of mPGES-1 inhibitors, using a phenotype-based assay involving image analyses. Seven primary hits among 49 compounds targeting mPGES-1 exhibited anti-influenza activities against A/Puerto Rico/8/1934 (H1N1) in a dose-dependent manner. The most effective hit, MPO-0047, suppressed influenza-induced p38 mitogen-activated protein kinase (MAPK), and c-Jun N-terminal kinase (JNK) activation. We also showed that mRNA levels of TNF-α, IL-8, CCL5/RANTES, and CXCL10/IP-10 were significantly reduced by the treatment of influenza-infected cells with MPO-0047. Exogenous PGE2 reversed the inhibitory effects of MPO-0047. Our results showed that this selective mPGES-1 inhibitor has anti-influenza effects by inhibiting PGE2 production, which suppresses the induction of pro-inflammatory genes. Taken together our data revealed that mPGES-1 inhibitor has the potential for further development as an influenza therapeutic agent.

Functional stability of 3D8 scFv, a nucleic acid-hydrolyzing single chain antibody, under different biochemical and physical conditions

3D8 single-chain Fv (scFv) is a catalytic nucleic acid antibody with anti-viral activity against a broad spectrum of viruses. Here we investigated the functional stability of 3D8 scFv to provide a basis for engineering a 3D8 scFv derivative and for developing stable formulations with improved stability and potential use as an anti-viral agent. The stability of 3D8 scFv was assessed by measuring its DNA-hydrolyzing activity under different biochemical and physical conditions using a fluorescence resonance energy transfer (FRET)-based method. In addition, the anti-influenza (H9N2) effect of 3D8 scFv was evaluated in A549 cells. 3D8 scFv was stable at 50°C for 6h at pH 7.2, for 3 days at pH 4–10 at 37°C and 30 days at pH 4–8 at 37°C. The stability was not affected by a reducing condition, freeze–thawing for up to 30 cycles, or lyophilization. Evaluation of the anti-virus effect showed that cells treated with 32–128 units of 3D8 scFv showed a 50% decrease in influenza replication compared to untreated cells. Based on its enzymatic stability in various biochemical and physical environments, 3D8 scFv holds good potential for development as an anti-viral therapeutic.

Lariciresinol-4-O-β-D-glucopyranoside from the root of Isatis indigotica inhibits influenza A virus-induced pro-inflammatory response

Ethnopharmacological relevanceIsatis indigotica is a traditional Chinese medicine. Its dried roots named “ban lan gen” in Chinese, are used for clinical treatment of virus infection, tumor, inflammation with a long history. However, its anti-influenza active ingredient and the underlying mechanism remain unclear. In this study, the anti-influenza and anti-inflammatory effects of a lignan glycoside: lariciresinol-4-O-β-D-glucopyranoside isolated from the root of I. indigotica on human alveolar epithelial cell line A549 infected with influenza A virus were investigated. Materials and methodsChemical and spectroscopic methods were employed to identify the structure of the lignan glycoside. Cytotoxicity of the lignan glycoside was analyzed using methylthiazolyltetrazolium (MTT) assay. The inhibitory activity against influenza virus of the lignan was determined by CPE inhibition assay. HEK-293 cells stably co-transfected with NF-κB responsive firefly luciferase and constitutively expressing GFP were employed for monitoring the effect of the lignan on NF-κB signal pathway activation. Nuclear export of viral ribonucleoprotein (RNP) complexes was monitored by indirect immunofluorescence. Quantitative real-time PCR was used to quantify the expression profiling of cytokines and chemokines after infection with influenza virus. ResultsWe showed that the lignan glycoside treatment was effective against the influenza A virus-induced cytopathic effect (CPE) in MDCK cells. Further study demonstrated the lignan glycoside attenuated virus-induced NF-κB activation, but did not affect export of viral ribonucleoprotein (RNP) complexes from the nucleus in late stages of infection. We revealed that the lignan glycoside suppressed influenza A virus (H1N1)-induced expression of the pro-inflammatory molecules IL-6, TNF-α, IL-8, MCP-1, IP-10 and IFN-α. Moreover, the cytokines and chemokines profiles induced by H9N2 virus resembled those of influenza virus H1N1, but the lignan glycoside reduced the expression of IP-10 and TNF-α. ConclusionsOur results suggest that the lignan glycoside is a bioactive component of I. indigotica which may contribute an adjunct to pharmacotherapy for influenza virus infection.