In vitro effects of bufotenine against RNA and DNA viruses.

Bufotenine, an alkaloid that can be found in plant extracts and skin secretions of amphibians, is reported to have potential antiviral activity. The present study evaluated the antiviral activity of bufotenine against different genetic lineages of rabies virus (RABV, a single-stranded, negative-sense RNA virus), canine coronavirus (CCoV, a positive-sense RNA virus) and two double-stranded DNA viruses (two strains of herpes simplex virus type 1/HSV-1 [KOS and the acyclovir-resistant HSV-1 strain 29R] and canine adenovirus 2, CAV-2). The maximal non-toxic bufotenine concentrations in Vero and BHK-21 cells were determined by MTT assays. The antiviral activity of bufotenine against each virus was assessed by examination of reductions in infectious virus titres and plaque assays. All experiments were performed with and without bufotenine, and the results were compared. Bufotenine demonstrated significant RABV inhibitory activity. No antiviral action was observed against CCoV, CAV-2 or HSV-1. These findings indicate that the antiviral activity of bufotenine is somewhat linked to the particular infectious dose used and the genetic lineage of the virus, although the mechanisms of its effects remain undetermined.

Antiviral Activity Exerted by Natural Products against Human Viruses.

Viral infections are responsible for several chronic and acute diseases in both humans and animals. Despite the incredible progress in human medicine, several viral diseases, such as acquired immunodeficiency syndrome, respiratory syndromes, and hepatitis, are still associated with high morbidity and mortality rates in humans. Natural products from plants or other organisms are a rich source of structurally novel chemical compounds including antivirals. Indeed, in traditional medicine, many pathological conditions have been treated using plant-derived medicines. Thus, the identification of novel alternative antiviral agents is of critical importance. In this review, we summarize novel phytochemicals with antiviral activity against human viruses and their potential application in treating or preventing viral disease.

Antiviral Activities of a Medicinal Plant Extract Against Sacbrood Virus in Honeybees.

BACKGROUND: Sacbrood is an infectious disease of the honey bee caused by Scbrood virus (SBV) which belongs to the family Iflaviridae and is especially lethal for Asian honeybee Apis cerana. Chinese Sacbrood virus (CSBV) is a geographic strain of SBV. Currently, there is a lack of an effective antiviral agent for controlling CSBV infection in honey bees. METHODS: Here, we explored the antiviral effect of a Chinese medicinal herb Radix isatidis on CSBV infection in A. cerana by inoculating the 3rd instar larvae with purified CSBV and treating the infected bee larvae with R. isatidis extract at the same time. The growth, development, and survival of larvae between the control and treatment groups were compared. The CSBV copy number at the 4th instar, 5th instar, and 6th instar larvae was measured by the absolute quantification PCR method. RESULTS: Bioassays revealed that R. isatidis extract significantly inhibited the replication of CSBV, mitigated the impacts of CSBV on larval growth and development, reduced the mortality of CSBV-infected A. cerana larvae, and modulated the expression of immune transcripts in infected bees. CONCLUSION: Although the mechanism underlying the inhibition of CSBV replication by the medicine plant will require further investigation, this study demonstrated the antiviral activity of R. isatidis extract and provides a potential strategy for controlling SBV infection in honey bees.

Ivermectin also inhibits the replication of bovine respiratory viruses (BRSV, BPIV-3, BoHV-1, BCoV and BVDV) in vitro.

Bovine respiratory disease (BRD) complex is an important viral infection that causes huge economic losses in cattle herds worldwide. However, there is no directly effective antiviral drug application against respiratory viral pathogens; generally, the metaphylactic antibacterial drug applications are used for BRD. Ivermectin (IVM) is currently used as a broad-spectrum anti-parasitic agent both for veterinary and human medicine on some occasions. Moreover, since it is identified as an inhibitor for importin α/ß-mediated nuclear localization signal (NLS), IVM is also reported to have antiviral potential against several RNA and DNA viruses. Since therapeutic use of IVM in COVID-19 cases has recently been postulated, the potential antiviral activity of IVM against bovine respiratory viruses including BRSV, BPIV-3, BoHV-1, BCoV and BVDV are evaluated in this study. For these purposes, virus titration assay was used to evaluate titers in viral harvest from infected cells treated with non-cytotoxic IVM concentrations (1, 2.5 and 5 µM) and compared to titers from non-treated infected cells. This study indicated that IVM inhibits the replication of BCoV, BVDV, BRSV, BPIV-3 and BoHV-1 in a dose-dependent manner in vitro as well as number of extracellular infectious virions. In addition, it was demonstrated that IVM has no clear effect on the attachment and penetration steps of the replication of the studied viruses. Finally, this study shows for the first time that IVM can inhibit infection of BRD-related viral agents namely BCoV, BPIV-3, BVDV, BRSV and BoHV-1 at the concentrations of 2.5 and 5 µM. Consequently, IVM, which is licensed for antiparasitic indications, also deserves to be evaluated as a broad-spectrum antiviral in BRD cases caused by viral pathogens.

In vitro virucidal activity of Echinaforce®, an Echinacea purpurea preparation, against coronaviruses, including common cold coronavirus 229E and SARS-CoV-2.

BACKGROUND: Coronaviruses (CoVs) were long thought to only cause mild respiratory and gastrointestinal symptoms in humans but outbreaks of Middle East Respiratory Syndrome (MERS)-CoV, Severe Acute Respiratory Syndrome (SARS)-CoV-1, and the recently identified SARS-CoV-2 have cemented their zoonotic potential and their capacity to cause serious morbidity and mortality, with case fatality rates ranging from 4 to 35%. Currently, no specific prophylaxis or treatment is available for CoV infections. Therefore we investigated the virucidal and antiviral potential of Echinacea purpurea (Echinaforce®) against human coronavirus (HCoV) 229E, highly pathogenic MERS- and SARS-CoVs, as well as the newly identified SARS-CoV-2, in vitro. METHODS: To evaluate the antiviral potential of the extract, we pre-treated virus particles and cells and evaluated remaining infectivity by limited dilution. Furthermore, we exposed cells to the extract after infection to further evaluate its potential as a prophylaxis and treatment against coronaviruses. We also determined the protective effect of Echinaforce® in re-constituted nasal epithelium. RESULTS: In the current study, we found that HCoV-229E was irreversibly inactivated when exposed to Echinaforce® at 3.2 µg/ml IC50. Pre-treatment of cell lines, however, did not inhibit infection with HCoV-229E and post-infection treatment had only a marginal effect on virus propagation at 50 µg/ml. However, we did observe a protective effect in an organotypic respiratory cell culture system by exposing pre-treated respiratory epithelium to droplets of HCoV-229E, imitating a natural infection. The observed virucidal activity of Echinaforce® was not restricted to common cold coronaviruses, as both SARS-CoV-1 and MERS-CoVs were inactivated at comparable concentrations. Finally, the causative agent of COVID-19, SARS-CoV-2 was also inactivated upon treatment with 50µg/ml Echinaforce®. CONCLUSIONS: These results show that Echinaforce® is virucidal against HCoV-229E, upon direct contact and in an organotypic cell culture model. Furthermore, MERS-CoV and both SARS-CoV-1 and SARS-CoV-2 were inactivated at similar concentrations of the extract. Therefore we hypothesize that Echinacea purpurea preparations, such as Echinaforce®, could be effective as prophylactic treatment for all CoVs due to their structural similarities.

Novel and potent inhibitors targeting DHODH are broad-spectrum antivirals against RNA viruses including newly-emerged coronavirus SARS-CoV-2.

Emerging and re-emerging RNA viruses occasionally cause epidemics and pandemics worldwide, such as the on-going outbreak of the novel coronavirus SARS-CoV-2. Herein, we identified two potent inhibitors of human DHODH, S312 and S416, with favorable drug-likeness and pharmacokinetic profiles, which all showed broad-spectrum antiviral effects against various RNA viruses, including influenza A virus, Zika virus, Ebola virus, and particularly against SARS-CoV-2. Notably, S416 is reported to be the most potent inhibitor so far with an EC50 of 17 nmol/L and an SI value of 10,505.88 in infected cells. Our results are the first to validate that DHODH is an attractive host target through high antiviral efficacy in vivo and low virus replication in DHODH knock-out cells. This work demonstrates that both S312/S416 and old drugs (Leflunomide/Teriflunomide) with dual actions of antiviral and immuno-regulation may have clinical potentials to cure SARS-CoV-2 or other RNA viruses circulating worldwide, no matter such viruses are mutated or not.

Antiviral and Virucidal Activities of Umesu Phenolics on Influenza Viruses.

In this study, umesu phenolics were purified from the salt extracts of Japanese apricot (Nanko-mume cultivar of Prunus mume Sieb. et Zucc.). Characterization of umesu phenolics revealed that, when added to the culture media of the infected cells, they inhibited the multiplication of influenza and many other RNA and DNA viruses. In addition to these antiviral activities, the phenolics significantly decreased the plating efficiency of influenza virus, if present in the virus inoculum. More drastic effects were observed in terms of virucidal activity; the infectivity of several strains of influenza viruses decreased less than 0.001 when they were incubated with 4 mg/ml phenolics at 30 ℃ for 5 min. The virucidal activity of phenolics was found to be more remarkable in acidic conditions; however, the activity was not merely a result of the acidity of the phenolics. These results clearly support the antiviral and virucidal activities of the umesu phenolics against influenza viruses and suggest their potential pharmacological usefulness as disinfectants or preventive medicine against superficial infections, such as the respiratory infections.

Inhibiting pyrimidine biosynthesis impairs Ebola virus replication through depletion of nucleoside pools and activation of innate immune responses.

Specific host pathways that may be targeted therapeutically to inhibit the replication of Ebola virus (EBOV) and other emerging viruses remain incompletely defined. A screen of 200,000 compounds for inhibition of an EBOV minigenome (MG) assay that measures the function of the viral polymerase complex identified as hits several compounds with an amino-tetrahydrocarbazole scaffold. This scaffold was structurally similar to GSK983, a compound previously described as having broad-spectrum antiviral activity due to its impairing de novo pyrimidine biosynthesis through inhibition of dihydroorotate dehydrogenase (DHODH). We generated compound SW835, the racemic version of GSK983 and demonstrated that SW835 and brequinar, another DHODH inhibitor, potently inhibit the MG assay and the replication of EBOV, vesicular stomatitis virus (VSV) and Zika (ZIKV) in vitro. Nucleoside and deoxynucleoside supplementation studies demonstrated that depletion of pyrimidine pools contributes to antiviral activity of these compounds. As reported for other DHODH inhibitors, SW835 and brequinar also induced expression of interferon stimulated genes (ISGs). ISG induction was demonstrated to occur without production of IFNα/ß and independently of the IFNα receptor and was not blocked by EBOV-encoded suppressors of IFN signaling pathways. Furthermore, we demonstrated that transcription factor IRF1 is required for this ISG induction, and that IRF1 induction requires the DNA damage response kinase ATM. Therefore, de novo pyrimidine biosynthesis is critical for the replication of EBOV and other RNA viruses and inhibition of this pathway activates an ATM and IRF1-dependent innate immune response that subverts EBOV immune evasion functions.

Nucleosides for the treatment of respiratory RNA virus infections.

Influenza virus, respiratory syncytial virus, human metapneumovirus, parainfluenza virus, coronaviruses, and rhinoviruses are among the most common viruses causing mild seasonal colds. These RNA viruses can also cause lower respiratory tract infections leading to bronchiolitis and pneumonia. Young children, the elderly, and patients with compromised cardiac, pulmonary, or immune systems are at greatest risk for serious disease associated with these RNA virus respiratory infections. In addition, swine and avian influenza viruses, together with severe acute respiratory syndrome-associated and Middle Eastern respiratory syndrome coronaviruses, represent significant pandemic threats to the general population. In this review, we describe the current medical need resulting from respiratory infections caused by RNA viruses, which justifies drug discovery efforts to identify new therapeutic agents. The RNA polymerase of respiratory viruses represents an attractive target for nucleoside and nucleotide analogs acting as inhibitors of RNA chain synthesis. Here, we present the molecular, biochemical, and structural fundamentals of the polymerase of the four major families of RNA respiratory viruses: Orthomyxoviridae, Pneumoviridae/Paramyxoviridae, Coronaviridae, and Picornaviridae. We summarize past and current efforts to develop nucleoside and nucleotide analogs as antiviral agents against respiratory virus infections. This includes molecules with very broad antiviral spectrum such as ribavirin and T-705 (favipiravir), and others targeting more specifically one or a few virus families. Recent advances in our understanding of the structure(s) and function(s) of respiratory virus polymerases will likely support the discovery and development of novel nucleoside analogs.

Antiviral activity of the "Virus Blocking Factor" (VBF) derived i.a. from Pelargonium extract and Sambucus juice against different human-pathogenic cold viruses in vitro.

THE AIM: The in-vitro antiviral activity of the "Virus Blocking Factor" (VBF), a combination of Pelargonium extract and Sambucus juice with addition of Betaglucan 1,3 / 1,6, Zincum gluconium, Acidum ascorbicum, was studied against human pathogenic viruses: Influenza A H1N1 (FluA H1N1), Rhinovirus B subtype 14 (HRV14), Respiratory Syncytial Virus (RSV), Parainfluenzavirus subtype 3 (Para 3), and Adenovirus C subtype 5 (Adeno 5). METHOD: Antiviral activity was assessed using plaque-reduction assays after adding the test substance post infection of the MDCK, HeLa and HEp-2 cells with the viruses. Ribavirin Virazol and - in case of Adenovirus an internal laboratory standard - were used as positive controls. Cytotoxic effects of VBF and VBF Control onto the virus permissive MDCK, HeLa and HEp-2 cells were examined. Non-toxic concentrations of VBF were determined by the Methylthiazoletetrazolium test (MTT-Test). RESULTS AND CONCLUSIONS: In all antiviral studies VBF showed (2.1%) a dose-dependent antiviral activity against FluA H1N1 and HRV14 at non-toxic concentrations. A very strong effect was demonstrated in concentrations of 2.5% and 1.25% where replication of H1N1 and HRV14 was nearly completely blocked. Dose-dependent antiviral activity was detectable against RSV in a concentration range of 1.25% to 0.63% of the test item. Due to toxic side effects of a 2.5% concentration at least a "minor effect" of about 30% (1.25% solution) against Para 3 infected HEp-2 cells could be determined. Concerning Adeno 5 not any antiviral activity could be demonstrated in all studies with all tested substance concentrations of VBF. VBF Control did not show any cytotoxicity and antiviral effects. Further research is needed to elucidate clinical effect of VBF.

Toward the discovery of dual HCMV-VZV inhibitors: Synthesis, structure activity relationship analysis, and cytotoxicity studies of long chained 2-uracil-3-yl-N-(4-phenoxyphenyl)acetamides.

The need for novel therapeutic options to fight herpesvirus infections still persists. Herein we report the design, synthesis and antiviral evaluation of a new family of non-nucleoside antivirals, derived from 1-[ω-(4-bromophenoxy)alkyl]uracil derivatives--previously reported inhibitors of human cytomegalovirus (HCMV). Introduction of the N-(4-phenoxyphenyl)acetamide side chain at N(3) increased their potency and widened activity spectrum. The most active compounds in the series exhibit submicromolar activity against different viral strains of HCMV and varicella zoster virus (VZV) replication in HEL cell cultures. Inactivity against other DNA and RNA viruses, including herpes simplex virus 1/2, points to a novel mechanism of antiviral action.

High-content image-based screening of a signal transduction pathway inhibitor small-molecule library against highly pathogenic RNA viruses.

High-content image-based screening was developed as an approach to test a small-molecule library of compounds targeting signal transduction pathways for antiviral activity against multiple highly pathogenic RNA viruses. Of the 2843 compounds screened, 120 compounds exhibited ≥60% antiviral activity. Four compounds (E225-0969, E528-0039, G118-0778, and G544-0735), which were most active against Rift Valley fever virus (RVFV) and showed broad-spectrum antiviral activity, were selected for further evaluation for their concentration-response profile and cytotoxicity. These compounds did not show any visible cytotoxicity at the highest concentration of compound tested (200 µM). All four of these compounds were more active than ribavirin against several viruses. One compound, E225-0969, had the lowest effective concentration (EC50 = 1.9-8.92 µM) for all the viruses tested. This compound was 13- and 43-fold more inhibitory against RVFV and Chikungunya virus (CHIKV), respectively, than ribavirin. The highest selectivity index (>106.2) was for E225-0969 against CHIKV. Time-of-addition assays suggested that all four lead compounds targeted early steps in the viral life cycle (entry and/or replication) but not virus egress. Overall, this work demonstrates that high-content image analysis can be used to screen chemical libraries for new antivirals against highly pathogenic viruses.

Design, synthesis, antiviral and cytostatic activity of ω-(1H-1,2,3-triazol-1-yl)(polyhydroxy)alkylphosphonates as acyclic nucleotide analogues.

The efficient synthesis of a new series of polyhydroxylated dibenzyl ω-(1H-1,2,3-triazol-1-yl)alkylphosphonates as acyclic nucleotide analogues is described starting from dibenzyl ω-azido(polyhydroxy)alkylphosphonates and selected alkynes under microwave irradiation. Selected O,O-dibenzylphosphonate acyclonucleotides were transformed into the respective phosphonic acids. All compounds were evaluated in vitro for activity against a broad variety of DNA and RNA viruses and for cytostatic activity against murine leukemia L1210, human T-lymphocyte CEM and human cervix carcinoma HeLa cells. Compound (1S,2S)-16b exhibited antiviral activity against Influenza A H3N2 subtype (EC50=20µM-visual CPE score; EC50=18µM-MTS method; MCC >100µM, CC50 >100µM) in Madin Darby canine kidney cell cultures (MDCK), and (1S,2S)-16k was active against vesicular stomatitis virus and respiratory syncytial virus in HeLa cells (EC50=9 and 12µM, respectively). Moreover, compound (1R,2S)-16l showed activity against both herpes simplex viruses (HSV-1, HSV-2) in HEL cell cultures (EC50=2.9 and 4µM, respectively) and feline herpes virus in CRFK cells (EC50=4µM) but at the same time it exhibited cytotoxicity toward uninfected cell (MCC⩾4µM). Several other compounds have been found to inhibit proliferation of L1210, CEM as well as HeLa cells with IC50 in the 4-50µM range. Among them compounds (1S,2S)- and (1R,2S)-16l were the most active (IC50 in the 4-7µM range).

An interferon-beta promoter reporter assay for high throughput identification of compounds against multiple RNA viruses.

Virus infection of host cells is sensed by innate pattern recognition receptors (PRRs) and induces production of type I interferons (IFNs) and other inflammatory cytokines. These cytokines orchestrate the elimination of the viruses but are occasionally detrimental to the hosts. The outcomes and pathogenesis of viral infection are largely determined by the specific interaction between the viruses and their host cells. Therefore, compounds that either inhibit viral infection or modulate virus-induced cytokine response should be considered as candidates for managing virus infection. The aim of the study was to identify compounds in both categories, using a single cell-based assay. Our screening platform is a HEK293 cell-based reporter assay where the expression of a firefly luciferase is under the control of a human IFN-ß promoter. We have demonstrated that infection of the reporter cell line with a panel of RNA viruses activated the reporter gene expression that correlates quantitatively with the levels of virus replication and progeny virus production, and could be inhibited in a dose-dependent manner by known antiviral compound or inhibitors of PRR signal transduction pathways. Using Dengue virus as an example, a pilot screening of a small molecule library consisting of 26,900 compounds proved the concept that the IFN-ß promoter reporter assay can serve as a convenient high throughput screening platform for simultaneous discovery of antiviral and innate immune response modulating compounds. A representative antiviral compound from the pilot screening, 1-(6-ethoxybenzo[d]thiazol-2-yl)-3-(3-methoxyphenyl) urea, was demonstrated to specifically inhibit several viruses belonging to the family of flaviviridae.

Biological evaluation of endophytic fungus, Chaetomium globosum JN711454, as potential candidate for improving drug discovery.

The main objective of this research work focused on investigating the biological and chemical aspects of endophytic fungus Chaetomium globosum, for pharmaceutical purposes to improve the drug discovery process. The endophytic C. globosum was isolated from healthy leaves of Egyptian medicinal plant Adiantum capillus-veneris collected from Saint Katherine Protectorate, Sinai, Egypt. The identification of C. globosum was on the basis of classical and molecular taxonomy. Gene encoding for 18S rRNA was partially sequenced, submitted to the GenBank and got the accession number JN711454, to resolve the phylogenetic relations with fungal ancestor using phylogenetic tree. To explore the biosynthetic power of endophytic C. globosum JN711454, the fungus was cultivated over five different media, oatmeal, rice, yeast malt glucose, potato dextrose agar (PDA) and Czapek's dox media, for 3 weeks at 30 °C, followed by extraction with different solvents, ethyl acetate (EA), and methanol. The ethyl acetate extract of C. globosum cultivated on PDA medium was the most potent extract. It showed strong antioxidant activity with EC50 11.5 µg/ml, potent anticancer activity with 55 % toxicity toward HepG-2 cells at 100 µg/ml and 66 % cytotoxicity to FGC4 cells at 250 µg/ml, promising butyrylcholinesterase inhibitory activities (>85 %), and moderate antimicrobial and stopped the attachment of HSV-2 virus to VERO cells. The metabolomic profiling of PDA-EA extract using LC-MS revealed the presence of several metabolites to which the observed bioactivities could be attributed. Here we report for the first time inhibitory activity of endophytic C. globosum JN711454 secondary metabolites to butyrylcholinesterase, one of neuro hydrolase enzymes that play a major role in development of Alzheimer's disease.

Cutthroat trout virus as a surrogate in vitro infection model for testing inhibitors of hepatitis E virus replication.

Hepatitis E virus (HEV) is one of the most important causes of acute hepatitis worldwide. Although most infections are self-limiting, mortality is particularly high in pregnant women. Chronic infections can occur in transplant and other immune-compromised patients. Successful treatment of chronic hepatitis E has been reported with ribavirin and pegylated interferon-alpha, however severe side effects were observed. We employed the cutthroat trout virus (CTV), a non-pathogenic fish virus with remarkable similarities to HEV, as a potential surrogate for HEV and established an antiviral assay against this virus using the Chinook salmon embryo (CHSE-214) cell line. Ribavirin and the respective trout interferon were found to efficiently inhibit CTV replication. Other known broad-spectrum inhibitors of RNA virus replication such as the nucleoside analog 2'-C-methylcytidine resulted only in a moderate antiviral activity. In its natural fish host, CTV levels largely fluctuate during the reproductive cycle with the virus detected mainly during spawning. We wondered whether this aspect of CTV infection may serve as a surrogate model for the peculiar pathogenesis of HEV in pregnant women. To that end the effect of three sex steroids on in vitro CTV replication was evaluated. Whereas progesterone resulted in marked inhibition of virus replication, testosterone and 17ß-estradiol stimulated viral growth. Our data thus indicate that CTV may serve as a surrogate model for HEV, both for antiviral experiments and studies on the replication biology of the Hepeviridae.

In vitro antiviral activity of plant extracts from Asteraceae medicinal plants.

BACKGROUND: Due to the high prevalence of viral infections having no specific treatment and the constant appearance of resistant viral strains, the development of novel antiviral agents is essential. The aim of this study was to evaluate the antiviral activity against bovine viral diarrhea virus, herpes simplex virus type 1 (HSV-1), poliovirus type 2 (PV-2) and vesicular stomatitis virus of organic (OE) and aqueous extracts (AE) from: Baccharis gaudichaudiana, B. spicata, Bidens subalternans, Pluchea sagittalis, Tagetes minuta and Tessaria absinthioides. A characterization of the antiviral activity of B. gaudichaudiana OE and AE and the bioassay-guided fractionation of the former and isolation of one active compound is also reported. METHODS: The antiviral activity of the OE and AE of the selected plants was evaluated by reduction of the viral cytopathic effect. Active extracts were then assessed by plaque reduction assays. The antiviral activity of the most active extracts was characterized by evaluating their effect on the pretreatment, the virucidal activity and the effect on the adsorption or post-adsorption period of the viral cycle. The bioassay-guided fractionation of B. gaudichaudiana OE was carried out by column chromatography followed by semipreparative high performance liquid chromatography fractionation of the most active fraction and isolation of an active compound. The antiviral activity of this compound was also evaluated by plaque assay. RESULTS: B. gaudichaudiana and B. spicata OE were active against PV-2 and VSV. T. absinthioides OE was only active against PV-2. The corresponding three AE were active against HSV-1. B. gaudichaudiana extracts (OE and AE) were the most selective ones with selectivity index (SI) values of 10.9 (PV-2) and > 117 (HSV-1). For this reason, both extracts of B. gaudichaudiana were selected to characterize their antiviral effects. Further bioassay-guided fractionation of B. gaudichaudiana OE led to an active fraction, FC (EC50 = 3.1 µg/ml; SI = 37.9), which showed antiviral activity during the first 4 h of the viral replication cycle of PV-2 and from which the flavonoid apigenin (EC50 = 12.2 ± 3.3 µM) was isolated as a major compound. CONCLUSIONS: The results showed that, among the species studied, B. gaudichaudiana seemed to be the most promising species as a source of antiviral agents.

A cell-based screening system for influenza A viral RNA transcription/replication inhibitors.

Although two classes of antivirals, NA inhibitors and M2 ion channel blockers, are licensed for influenza treatment, dual resistant mutants, including highly pathogenic H5N1 viruses, have appeared. Alternative treatment options are, therefore, needed. Influenza A viral RNA (vRNA) transcription/replication is a promising target for antiviral development, since it is essential for virus replication. Accordingly, an efficient and reliable method to identify vRNA transcription/replication inhibitors is desirable. Here, we developed a cell-based screening system by establishing a cell line that stably expresses influenza viral ribonucleoprotein complex (vRNP). Compound library screening using this cell line allowed us to identify a compound that inhibits vRNA transcription/replication by using reporter protein expression from virus-like RNA as a readout and virus replication in vitro. vRNP-expressing cells have potential as a simple and convenient high-throughput screening (HTS) system, and, thus, are promising to identify vRNA transcription/replication inhibitors for various RNA viruses, especially for primary screens.

Novel 1-acyl-4-substituted semicarbazide derivatives of primaquine - synthesis, cytostatic, antiviral and antioxidative studies.

A series of novel 1,4-substituted semicarbazides 5a-g with a primaquine moiety bridged by a carbonyl group at position 1 and a cycloalkyl, aryl, benzyloxy or hydroxy substituent at position 4 were prepared and biologically evaluated. The synthetic pathways applied for preparation of the title compounds involved benzotriazole as synthetic auxiliary. Primaquine semicarbazides 5a-g and their synthetic precursors benzotriazolecarbonyl semicarbazides 4 were evaluated for cytostatic, antiviral and antioxidative activities. All compounds of the series 5 showed high selectivity towards MCF-7 cells (breast carcinoma) with IC(50) values in the low micromolar range and the most active was benzyl derivative 5c (IC(50) 1 ± 0.2 µM). The benzhydryl derivative 5e showed significant cytostatic activities towards all the tested cell lines (IC(50) 4-18 µM). The same compound was the strongest lipoxygenase inhibitor as well (51%). The highest antioxidant activity was demonstrated for the hydroxy derivative 5g and benzotriazolecarbonyl semicarbazides 4b,c (61.2-68.5%). No antiviral activity was observed against a wide variety of DNA and RNA viruses.

Antiviral activity in vitro of two preparations of the herbal medicinal product Sinupret® against viruses causing respiratory infections.

Sinupret(®), a herbal medicinal product made from Gentian root, Primula flower, Elder flower, Sorrel herb, and Verbena herb is frequently used in the treatment of acute and chronic rhinosinusitis and respiratory viral infections such as common cold. To date little is known about its potential antiviral activity. Therefore experiments have been performed to measure the antiviral activity of Sinupret(®) oral drops (hereinafter referred to as "oral drops") and Sinupret(®) dry extract (hereinafter referred to as "dry extract"), in vitro against a broad panel of both enveloped and non-enveloped human pathogenic RNA and DNA viruses known to cause infections of the upper respiratory tract: influenza A, Chile 1/83 (H1N1) virus (FluA), Porcine Influenza A/California/07/2009 (H1N1) virus (pFluA), parainfluenza type 3 virus (Para 3), respiratory syncytial virus, strain Long (RSV), human rhinovirus B subtype 14 (HRV 14), coxsackievirus subtype A9 (CA9), and adenovirus C subtype 5 (Adeno 5). Concentration-dependent antiviral activity (EC(50) between 13.8 and 124.8 µg/ml) of Sinupret(®) was observed against RNA as well as DNA viruses independent of a viral envelope. Remarkable antiviral activity was shown against Adeno 5, HRV 14 and RSV in which dry extract was significantly superior to oral drops. This could be ascertained with different assays as plaque-reduction assays in plaque forming units (PFU), the analyses of a cytopathogenic effect (CPE) and with enzyme immunoassays (ELISA) to determine the amount of newly synthesised virus. Our results demonstrate that Sinupret(®) shows a broad spectrum of antiviral activity in vitro against viruses commonly known to cause respiratory infections.