Human TBK1 deficiency leads to autoinflammation driven by TNF-induced cell death.

TANK binding kinase 1 (TBK1) regulates IFN-I, NF-κB, and TNF-induced RIPK1-dependent cell death (RCD). In mice, biallelic loss of TBK1 is embryonically lethal. We discovered four humans, ages 32, 26, 7, and 8 from three unrelated consanguineous families with homozygous loss-of-function mutations in TBK1. All four patients suffer from chronic and systemic autoinflammation, but not severe viral infections. We demonstrate that TBK1 loss results in hypomorphic but sufficient IFN-I induction via RIG-I/MDA5, while the system retains near intact IL-6 induction through NF-κB. Autoinflammation is driven by TNF-induced RCD as patient-derived fibroblasts experienced higher rates of necroptosis in vitro, and CC3 was elevated in peripheral blood ex vivo. Treatment with anti-TNF dampened the baseline circulating inflammatory profile and ameliorated the clinical condition in vivo. These findings highlight the plasticity of the IFN-I response and underscore a cardinal role for TBK1 in the regulation of RCD.

Inhibition of acid sphingomyelinase by ambroxol prevents SARS-CoV-2 entry into epithelial cells.

The acid sphingomyelinase/ceramide system has been shown to be important for cellular infection with at least some viruses, for instance, rhinovirus or severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Functional inhibition of the acid sphingomyelinase using tricyclic antidepressants prevented infection of epithelial cells, for instance with SARS-CoV-2. The structure of ambroxol, that is, trans-4-[(2,4-dibromanilin-6-yl)-methyamino]-cyclohexanol, a mucolytic drug applied by inhalation, suggests that the drug might inhibit the acid sphingomyelinase and thereby infection with SARS-CoV-2. To test this, we used vesicular stomatitis virus pseudoviral particles presenting SARS-CoV-2 spike protein on their surface (pp-VSV-SARS-CoV-2 spike), a bona fide system for mimicking SARS-CoV-2 entry into cells. Viral uptake and formation of ceramide localization were determined by fluorescence microscopy, activity of the acid sphingomyelinase by consumption of [14C]sphingomyelin and ceramide was quantified by a kinase method. We found that entry of pp-VSV-SARS-CoV-2 spike required activation of acid sphingomyelinase and release of ceramide, events that were all prevented by pretreatment with ambroxol. We also obtained nasal epithelial cells from human volunteers prior to and after inhalation of ambroxol. Inhalation of ambroxol reduced acid sphingomyelinase activity in nasal epithelial cells and prevented pp-VSV-SARS-CoV-2 spike-induced acid sphingomyelinase activation, ceramide release, and entry of pp-VSV-SARS-CoV-2 spike ex vivo. The addition of purified acid sphingomyelinase or C16 ceramide restored entry of pp-VSV-SARS-CoV-2 spike into ambroxol-treated epithelial cells. We propose that ambroxol might be suitable for clinical studies to prevent coronavirus disease 2019.

Chemogenetic ON and OFF switches for RNA virus replication.

Therapeutic application of RNA viruses as oncolytic agents or gene vectors requires a tight control of virus activity if toxicity is a concern. Here we present a regulator switch for RNA viruses using a conditional protease approach, in which the function of at least one viral protein essential for transcription and replication is linked to autocatalytical, exogenous human immunodeficiency virus (HIV) protease activity. Virus activity can be en- or disabled by various HIV protease inhibitors. Incorporating the HIV protease dimer in the genome of vesicular stomatitis virus (VSV) into the open reading frame of either the P- or L-protein resulted in an ON switch. Here, virus activity depends on co-application of protease inhibitor in a dose-dependent manner. Conversely, an N-terminal VSV polymerase tag with the HIV protease dimer constitutes an OFF switch, as application of protease inhibitor stops virus activity. This technology may also be applicable to other potentially therapeutic RNA viruses.

DDX3 inhibitors show antiviral activity against positive-sense single-stranded RNA viruses but not against negative-sense single-stranded RNA viruses: The coxsackie B model.

Picornaviridae are positive-sense single stranded RNA viruses with a similar genomic structure lacking a cap at the 5' end, but with a highly structured 5'-untranslated region (UTR) containing an internal ribosome entry site (IRES). IRES allows ribosomes to be recruited by the viral RNA and initiate translation in a cap-independent manner. Coxsackie virus type B (CV-B) belong to Picornaviridae and are widespread in human population. They usually cause subclinical infections but, occasionally, also severe diseases with various clinical manifestations. CV-B have no specific therapy. DEAD-box polypeptide 3 (DDX3) is a member of the Asp-Glu-Ala-Asp (DEAD)-box family with an ATP-dependent RNA unwinding helicase activity. Recently, several positive-sense single strand RNA viruses have been shown to need DDX3 for their translation. Here, we show that several DDX3 inhibitors reduced CV-B replication and production of viral protein, particularly when added within 12 h of infection. Based on in vitro and in silico data, we hypothesized that DDX3 inhibitors hamper interaction between DDX3 and viral IRES in a stereodynamic fashion. Accordingly, the DDX3 inhibitors tested have no activity against the Vesicular Stomatitis virus and Measles virus, which are negative-sense single stranded RNA viruses and use cap-dependent translation. This study suggests that DDX3 is required by RNA viruses lacking a cap and show that this enzyme is a valuable target to design antiviral molecules against CV-B. Thus, DDX3 is dispensable for cap-dependent translation, but required for translation of transcripts containing secondary structure in their UTRs.

MicroRNA-218 inhibits type I interferon production and facilitates virus immune evasion via targeting RIG-I.

The host protective immunity against viral infection requires the effective detection of viral antigens and the subsequent production of type I interferons (IFNs) by host immune cells. Retinoic acid-inducible gene I (RIG-I) is the crucial signaling element responsible for sensing viral RNA component and initiating the downstream antiviral signaling pathways, leading to the production of type I IFNs. In this work, we identified microRNA-218 (miR-218) as a new virus-induced miRNA that dampens the expression of RIG-I in mouse and human macrophages, leading to the impaired production of type I IFNs. Interfering miR-218 expression rescued RIG-I-mediated antiviral signaling and thus protected macrophages from viral infection. Hence, our results provide new understanding of miRNA-mediated viral immune evasion and may be potentially useful for the treatment of viral infection in the future.

Glucosamine promotes hepatitis B virus replication through its dual effects in suppressing autophagic degradation and inhibiting MTORC1 signaling.

Glucosamine (GlcN), a dietary supplement widely utilized to promote joint health and effective in the treatment of osteoarthritis, is an effective macroautophagy/autophagy activator in vitro and in vivo. Previous studies have shown that autophagy is required for hepatitis B virus (HBV) replication and envelopment. The objective of this study was to determine whether and how GlcN affects HBV replication, using in vitro and in vivo experiments. Our data demonstrated that HBsAg production and HBV replication were significantly increased by GlcN treatment. Confocal microscopy and western blot analysis showed that the amount of autophagosomes and the levels of autophagic markers MAP1LC3/LC3-II and SQSTM1 were clearly elevated by GlcN treatment. GlcN strongly blocked autophagic degradation of HBV virions and proteins by inhibiting lysosomal acidification through its amino group. Moreover, GlcN further promoted HBV replication by inducing autophagosome formation via feedback inhibition of mechanistic target of rapamycin kinase complex 1 (MTORC1) signaling in an RRAGA (Ras related GTP binding A) GTPase-dependent manner. In vivo, GlcN application promoted HBV replication and blocked autophagic degradation in an HBV hydrodynamic injection mouse model. In addition, GlcN promoted influenza A virus, enterovirus 71, and vesicular stomatitis virus replication in vitro. In conclusion, GlcN efficiently promotes virus replication by inducing autophagic stress through its dual effects in suppressing autophagic degradation and inhibiting MTORC1 signaling. Thus, there is a potential risk of enhanced viral replication by oral GlcN intake in chronically virally infected patients.Abbreviations: ACTB: actin beta; ATG: autophagy-related; CMIA: chemiluminescence immunoassay; ConA: concanavalin A; CQ: chloroquine; CTSD: cathepsin D; DAPI: 4',6-diamidino-2-phenylindole; EV71: enterovirus 71; GalN: galactosamine; GFP: green fluorescence protein; GlcN: glucosamine; GNPNAT1: glucosamine-phosphate N-acetyltransferase 1; HBP: hexosamine biosynthesis pathway; HBV: hepatitis B virus; HBcAg: hepatitis B core antigen; HBsAg: hepatitis B surface antigen; HBeAg: hepatitis B e antigen; HBV RI: hepatitis B replicative intermediate; IAV: influenza A virus; LAMP1: lysosomal associated membrane protein 1; LAMTOR: late endosomal/lysosomal adaptor, MAPK and MTOR activator; ManN: mannosamine; MAP1LC3/LC3: microtubule associated protein 1 light chain 3; MTORC1: mechanistic target of rapamycin kinase complex 1; PHH: primary human hepatocyte; RAB7: RAB7A, member RAS oncogene family; RPS6KB1: ribosomal protein S6 kinase B1; RRAGA: Ras related GTP binding A; RT-PCR: reverse transcriptase polymerase chain reaction; SEM: standard error of the mean; siRNA: small interfering RNA; SQSTM1/p62: sequestosome 1; UAP1: UDP-N-acetylglucosamine pyrophosphorylase 1; VSV: vesicular stomatitis virus.

Vesiculopolins, a New Class of Anti-Vesiculoviral Compounds, Inhibit Transcription Initiation of Vesiculoviruses.

Vesicular stomatitis virus (VSV) represents a promising platform for developing oncolytic viruses, as well as vaccines against significant human pathogens. To safely control VSV infection in humans, small-molecule drugs that selectively inhibit VSV infection may be needed. Here, using a cell-based high-throughput screening assay followed by an in vitro transcription assay, compounds with a 7-hydroxy-6-methyl-3,4-dihydroquinolin-2(1H)-one structure and an aromatic group at position 4 (named vesiculopolins, VPIs) were identified as VSV RNA polymerase inhibitors. The most effective compound, VPI A, inhibited VSV-induced cytopathic effects and in vitro mRNA synthesis with micromolar to submicromolar 50% inhibitory concentrations. VPI A was found to inhibit terminal de novo initiation rather than elongation for leader RNA synthesis, but not mRNA capping, with the VSV L protein, suggesting that VPI A is targeted to the polymerase domain in the L protein. VPI A inhibited transcription of Chandipura virus, but not of human parainfluenza virus 3, suggesting that it specifically acts on vesiculoviral L proteins. These results suggest that VPIs may serve not only as molecular probes to elucidate the mechanisms of transcription of vesiculoviruses, but also as lead compounds to develop antiviral drugs against vesiculoviruses and other related rhabdoviruses.

Half-life extension of porcine interferon-α by fusion to the IgG-binding domain of streptococcal G protein.

Recombinant interferon-α (rIFN-α) has been widely used for treating viral infections. However, the clinical efficacy of unmodified rIFN-α is limited due to small molecular size and rapid clearance from circulation. In this study we developed a novel strategy for half-life extension of porcine IFN-α (PoIFN-α) by fusion to the immunoglobulin (Ig)-binding C2 domain of streptococcal protein G (SPG). The coding sequences for PoIFN-α6 and SPG C2 domain, with a tobacco etch virus (TEV) protease recognition sequence introduced at the 5-end, were cloned into an elastin-like polypeptide (ELP) fusion expression vector and expressed as an ELP-PoIFNα-C2 fusion protein. After optimization of the conditions for soluble protein expression and purification, the fusion protein was purified to more than 90% purity by two rounds of inverse transition cycling (ITC) in the presence of 0.5% Triton X-100. After cleavage with self-aggregating peptide ELK-16-tagged tobacco etch virus protease, the protease was removed by quick centrifugation and PoIFNα-C2 protein was recovered by an additional round of ITC with 98% purity. Western blotting analysis showed that PoIFNα-C2 protein had the specific affinity for pig IgG binding. The antiviral assay showed that PoIFNα-C2 protein had potent antiviral activities against vesicular stomatitis virus and porcine pseudorabies virus. After single intravenous or subcutaneous injection into rats, PoIFNα-C2 protein showed 16- or 4-fold increase in serum half-life with significantly improved bioavailability.

Virucidal, antiviral and immunomodulatory activities of ß-escin and Aesculus hippocastanum extract.

OBJECTIVES: ß-Escin, one of the constituents of Aesculus hippocastanum L. (Hippocastanaceae) seed extract (AH), inhibits NF-κB activation, which plays an important role in HSV-1 replication. The aim was to examine the antiherpetic activity of ß-escin and AH, as well as their effect on the activation of NF-κB and AP-1 and cytokine secretion in epithelial cells and macrophages. METHODS: Cell viability was evaluated using MTT assay, and antiviral and virucidal activity was determined by plaque assay. The effect on NF-κB and AP-1 signalling pathways activation was determined by a luciferase reporter assay, and cytokine production was measured by ELISA. KEY FINDINGS: ß-Escin and AH had virucidal and anti-HSV-1 activities, and the antiviral activity was discovered for other enveloped viruses (VSV and Dengue). Moreover, ß-escin and AH significantly reduced NF-κB and AP-1 activation and cytokine production in macrophages stimulated with HSV-1 and TLRs ligands. However, an enhanced activation of these pathways and an increase in the levels of pro-inflammatory cytokines in ß-escin and AH-treated HSV-1-infected epithelial cells were found. CONCLUSIONS: This study demonstrates virucidal and broad-spectrum antiviral activities for ß escin and AH. Besides, ß-escin and AH modulate cytokine production depending on the stimuli (viral or non-viral) and the cell type under study.

Identification of a novel porcine OASL variant exhibiting antiviral activity.

2', 5'-Oligoadenylate synthetase-lilke (OASL) protein is an atypical oligoadenylate synthetase (OAS) family member, which possesses antiviral activity but lacks 2', 5'-oligoadenylate synthetase activity. Here, a novel variant of porcine OASL (pOASL2) was identified through RT-PCR amplification. This gene is distinguishable from the previously described wild-type porcine OASL (pOASL1). The gene appears to be derived from a truncation of exon 4 plus 8 nucleotides of exon 5 with a premature termination, measuring only 633 bp in length, although its position corresponds to that of pOASL1. Given this novel gene appears to be a variant of pOASL, we assayed for antiviral activity of the protein. We demonstrated that pOASL2 could inhibit Japanese encephalitis virus (JEV) proliferation as well as pOASL1 in a transient overexpression assay of pOASL1 and pOASL2 in PK-15 and Vero cells. In addition to JEV, pOASL1 and pOASL2 also decreased the proliferations of Porcine reproductive and respiratory syndrome virus (PRRSV) and vesicular stomatitis virus (VSV), but did not exhibit antiviral activity against pseudorabies virus (PRV). Structural analysis showed that the pOASL2 gene retained only the first three exons at the 5'-. To investigate the role of the αN4 helix in pOASL in antiviral responses like that in hOASL, we mutated key residues in the anchor domain of the αN4 helix in pOASL2, based on the domain's location in hOASL. However, the antiviral activity of pOASL2 was not affected. Thus, the αN4 helix of pOASL likely does not play a significant role in its antiviral activity. In conclusion, pOASL2 acts as a new splice isoform of pOASL that plays a role in resistance to infection of several kinds of RNA viruses.

Antiviral activity and metal ion-binding properties of some 2-hydroxy-3-methoxyphenyl acylhydrazones.

Here we report on the results obtained from an antiviral screening, including herpes simplex virus, vaccinia virus, vesicular stomatitis virus, Coxsackie B4 virus or respiratory syncytial virus, parainfluenza-3 virus, reovirus-1 and Punta Toro virus, of three 2-hydroxy-3-methoxyphenyl acylhydrazone compounds in three cell lines (i.e. human embryonic lung fibroblast cells, human cervix carcinoma cells, and African Green monkey kidney cells). Interesting antiviral EC50 values are obtained against herpes simplex virus-1 and vaccinia virus. The biological activity of acylhydrazones is often attributed to their metal coordinating abilities, so potentiometric and microcalorimetric studies are here discussed to unravel the behavior of the three 2-hydroxy-3-methoxyphenyl compounds in solution. It is worth of note that the acylhydrazone with the higher affinity for Cu(II) ions shows the best antiviral activity against herpes simplex and vaccinia virus (EC50 ~ 1.5 µM, minimal cytotoxic concentration = 60 µM, selectivity index = 40).

Antiviral Activity of Synthetic Peptides Derived from Physiological Proteins.

BACKGROUND/AIMS: New antivirals are needed to supplement or replace currently used drugs. The aim of this study was to evaluate the antiviral activity of synthetic peptides derived from physiological proteins. METHODS: Vero cell monolayers were infected with herpes simplex virus 1, vesicular stomatitis virus, adenovirus, and coxsackievirus B5 strains in the presence of different concentrations of the selected peptides and viral yield was determined by plaque reduction assays to evaluate the antiviral activity of the peptides. Virucidal activity was evaluated by determining the residual infectivity of viral suspensions treated for 1 h with the peptides at the same concentrations as in the viral yield assays. RESULTS: Among the investigated peptides, the killer peptide proved to exert a considerable antiviral activity against herpes simplex virus, attributable to a direct effect on virus particles, while its derivative K10S showed to be effective against the four investigated virus strains only at the highest concentration tested, yet, the inhibitory effects were only partial. CONCLUSION: Overall, initial evidence is provided on the antiviral activity of several peptides, as well as of their derivatives. Further investigation is warranted to ascertain the mechanism of action in order to develop new potential antiviral drugs.

Dense Granule Protein-7 (GRA-7) of Toxoplasma gondii inhibits viral replication in vitro and in vivo.

Dense granule protein-7 (GRA-7) is an excretory protein of Toxoplasma gondii. It is a potential serodiagnostic marker and vaccine candidate for toxoplasmosis. Previous reports demonstrated that GRA-7 induces innate immune responses in macrophages by interacting with TRAF6 via the MyD88-dependent pathway. In the present study, we evaluated the antiviral activity and induction of an antiviral state by GRA-7 both in vitro and in vivo. It was observed that GRA-7 markedly reduced the replication of vesicular stomatitis virus (VSV-GFP), influenza A virus (PR8-GFP), coxsackievirus (H3-GFP), herpes simplex virus (HSV-GFP), and adenovirus-GFP in epithelial (HEK293T/HeLa) and immune (RAW264.7) cells. These antiviral activities of GRA-7 were attributed to the induction of type I interferon (IFN) signaling, resulting in the secretion of IFNs and pro-inflammatory cytokines. Additionally, in BALB/c mice, intranasal administration of GRA-7 prevented lethal infection by influenza A virus (H1N1) and exhibited prophylactic effects against respiratory syncytial virus (RSV-GFP). Collectively, these results suggested that GRA-7 exhibits immunostimulatory and broad spectrum antiviral activities via type I IFN signaling. Thus, GRA-7 can be potentially used as a vaccine adjuvant or as a candidate drug with prophylactic potential against viruses.

Inhibition of highly pathogenic avian influenza (HPAI) virus by a peptide derived from vFLIP through its direct destabilization of viruses.

The antiviral activities of synthesized Kα2-helix peptide, which was derived from the viral FLICE-like inhibitor protein (vFLIP) of Kaposi's sarcoma-associated herpesvirus (KSHV), against influenza A virus (IAV) were investigated in vitro and in vivo, and mechanisms of action were suggested. In addition to the robust autophagy activity of the Kα2-helix peptide, the present study showed that treatment with the Kα2 peptide fused with the TAT peptide significantly inhibited IAV replication and transmission. Moreover, TAT-Kα2 peptide protected the mice, that were challenged with lethal doses of highly pathogenic influenza A H5N1 or H1N1 viruses. Mechanistically, we found that TAT-Kα2 peptide destabilized the viral membranes, depending on their lipid composition of the viral envelop. In addition to IAV, the Kα2 peptide inhibited infections with enveloped viruses, such as Vesicular Stomatitis Virus (VSV) and Respiratory Syncytial Virus (RSV), without cytotoxicity. These results suggest that TAT-Kα2 peptide is a potential antiviral agent for controlling emerging or re-emerging enveloped viruses, particularly diverse subtypes of IAVs.

Site-Specific Transglutaminase-Mediated Conjugation of Interferon α-2b at Glutamine or Lysine Residues.

Interferon α (IFN α) subtypes are important protein drugs that have been used to treat infectious diseases and cancers. Here, we studied the reactivity of IFN α-2b to microbial transglutaminase (TGase) with the aim of obtaining a site-specific conjugation of this protein drug. Interestingly, TGase allowed the production of two monoderivatized isomers of IFN with high yields. Characterization by mass spectrometry of the two conjugates indicated that they are exclusively modified at the level of Gln101 if the protein is reacted in the presence of an amino-containing ligand (i.e., dansylcadaverine) or at the level of Lys164 if a glutamine-containing molecule is used (i.e., carbobenzoxy-l-glutaminyl-glycine, ZQG). We explained the extraordinary specificity of the TGase-mediated reaction on the basis of the conformational features of IFN. Indeed, among the 10 Lys and 12 Gln residues of the protein, only Gln101 and Lys164 are located in highly flexible protein regions. The TGase-mediated derivatization of IFN was then applied to the production of IFN derivatives conjugated to a 20 kDa polyethylene glycol (PEG), using PEG-NH2 for Gln101 derivatization and PEG modified with ZQG for Lys164 derivatization. The two mono-PEGylated isomers of IFN were obtained in good yields, purified, and characterized in terms of protein conformation, antiviral activity, and pharmacokinetics. Both conjugates maintained a native-like secondary structure, as indicated by far-UV circular dichroism spectra. Importantly, they disclosed good in vitro antiviral activity retention (about only 1.6- to 1.8-fold lower than that of IFN) and half-lives longer (about 5-fold) than that of IFN after intravenous administration to rats. Overall, these results provide evidence that TGase can be used for the development of site-specific derivatives of IFN α-2b possessing interesting antiviral and pharmacokinetic properties.

Cloning, expression, and antiviral activity of interferon ß from the Chinese microbat, Myotis davidii.

Bats are natural reservoir hosts for many viruses that produce no clinical symptoms in bats. Therefore, bats may have evolved effective mechanisms to control viral replication. However, little information is available on bat immune responses to viral infection. Type I interferon (IFN) plays a key role in controlling viral infections. In this study, we report the cloning, expression, and biological activity of interferon ß (IFNß) from the Chinese microbat species, Myotis davidii. We demonstrated the upregulation of IFNB and IFN-stimulated genes in a kidney cell line derived from M. davidii after treatment with polyI:C or infection with Sendai virus. Furthermore, the recombinant IFNß inhibited vesicular stomatitis virus and bat adenovirus replication in cell lines from two bat species, M. davidii and Rhinolophus sinicus. We provide the first in vitro evidence of IFNß antiviral activity in microbats, which has important implications for virus interactions with these hosts.

Inhibition of viral protein translation by indomethacin in vesicular stomatitis virus infection: role of eIF2α kinase PKR.

Indomethacin, a cyclooxygenase-1 and -2 inhibitor widely used in the clinic for its potent anti-inflammatory/analgesic properties, possesses antiviral activity against several viral pathogens; however, the mechanism of antiviral action remains elusive. We have recently shown that indomethacin activates the double-stranded RNA (dsRNA)-dependent protein kinase R (PKR) in human colon cancer cells. Because of the important role of PKR in the cellular defence response against viral infection, herein we investigated the effect of indomethacin on PKR activity during infection with the prototype rhabdovirus vesicular stomatitis virus. Indomethacin was found to activate PKR in an interferon- and dsRNA-independent manner, causing rapid (< 5 min) phosphorylation of eukaryotic initiation factor-2 α-subunit (eIF2α). These events resulted in shutting off viral protein translation and blocking viral replication (IC50 = 2 µM) while protecting host cells from virus-induced damage. Indomethacin did not affect eIF2α kinases PKR-like endoplasmic reticulum-resident protein kinase (PERK) and general control non-derepressible-2 (GCN2) kinase, and was unable to trigger eIF2α phosphorylation in the presence of PKR inhibitor 2-aminopurine. In addition, small-interfering RNA-mediated PKR gene silencing dampened the antiviral effect in indomethacin-treated cells. The results identify PKR as a critical target for the antiviral activity of indomethacin and indicate that eIF2α phosphorylation could be a key element in the broad spectrum antiviral activity of the drug.

Epimedium koreanum Nakai displays broad spectrum of antiviral activity in vitro and in vivo by inducing cellular antiviral state.

Epimedium koreanum Nakai has been extensively used in traditional Korean and Chinese medicine to treat a variety of diseases. Despite the plant's known immune modulatory potential and chemical make-up, scientific information on its antiviral properties and mode of action have not been completely investigated. In this study, the broad antiviral spectrum and mode of action of an aqueous extract from Epimedium koreanum Nakai was evaluated in vitro, and moreover, the protective effect against divergent influenza A subtypes was determined in BALB/c mice. An effective dose of Epimedium koreanum Nakai markedly reduced the replication of Influenza A Virus (PR8), Vesicular Stomatitis Virus (VSV), Herpes Simplex Virus (HSV) and Newcastle Disease Virus (NDV) in RAW264.7 and HEK293T cells. Mechanically, we found that an aqueous extract from Epimedium koreanum Nakai induced the secretion of type I IFN and pro-inflammatory cytokines and the subsequent stimulation of the antiviral state in cells. Among various components present in the extract, quercetin was confirmed to have striking antiviral properties. The oral administration of Epimedium koreanum Nakai exhibited preventive effects on BALB/c mice against lethal doses of highly pathogenic influenza A subtypes (H1N1, H5N2, H7N3 and H9N2). Therefore, an extract of Epimedium koreanum Nakai and its components play roles as immunomodulators in the innate immune response, and may be potential candidates for prophylactic or therapeutic treatments against diverse viruses in animal and humans.

Attenuation of vesicular stomatitis virus infection of brain using antiviral drugs and an adeno-associated virus-interferon vector.

Vesicular stomatitis virus (VSV) shows promise as a vaccine-vector and oncolytic virus. However, reports of neurotoxicity of VSV remain a concern. We compared 12 antiviral compounds to control infection of VSV-CT9-M51 and VSV-rp30 using murine and human brain cultures, and in vivo mouse models. Inhibition of replication, cytotoxicity and infectivity was strongest with ribavirin and IFN-α and to some extent with mycophenolic acid, chloroquine, and adenine 9-ß-d-arabinofuranoside. To generate continuous IFN exposure, we made an adeno-associated virus vector expressing murine IFN; AAV-mIFN-ß protected mouse brain cells from VSV, as did a combination of IFN, ribavirin and chloroquine. Intracranial AAV-mIFN-ß protected the brain against VSV-CT9-M51. In SCID mice bearing human glioblastoma, AAV-mIFN-ß moderately enhanced survival. VSV-CT9-M51 doubled median survival when administered after AAV-mIFN-ß; some surviving mice showed complete tumor destruction. Together, these data suggest that AAV-IFN or IFN with ribavirin and chloroquine provide an optimal anti-virus combination against VSV in the brain.

In vitro biological characterization of IFN-ß-1a major glycoforms.

Recombinant human interferon ß-1a (IFN-ß-1a) is extensively used as the first-line treatment of relapsing forms of multiple sclerosis. Its glycosylation is recognized as having a complex impact on a wide range of molecule characteristics and functions. The present study reports the enrichment of IFN-ß-1a glycoforms and their physicochemical and biological characterization by means of electrospray ionization-mass spectrometry, sialic acid content, thermal denaturation and various in vitro bioassays (antiproliferative, antiviral, immunomodulatory and reporter gene assay). The glycoforms were fractionated by means of cation-exchange chromatography using recombinant IFN-ß-1a derived from Chinese Hamster Ovary cell culture as starting material. The obtained fractions contained bi- and higher-antennarity glycans as described in the European Pharmacopoeia monograph (Nr. 1639E, Interferon beta 1a concentrated solution). The in vitro bioassay responses revealed a correlation mainly with the glycan antennarity. It is therefore suggested that all glycoforms have biological activity and play a role in modulating the overall IFN-ß biological activity with higher-antennarity glycoforms being able to better sustain IFN-ß-1a bioactivity over time. These data indicate the role of IFN-ß-1a glycosylation in vivo and shed new light on the role of the glycosylation heterogeneity, in particular with regard to antennarity, on biological properties of glycoproteins.