<Emphasis Type="Italic">In Vitro</Emphasis> antiviral activity of red alga, <Emphasis Type="Italic">Polysiphonia morrowii</Emphasis> extract and its bromophenols against fish pathogenic infectious hematopoietic necrosis virus and infectious pancreatic necrosis virus

Our previous investigation revealed that 80% methanolic extract of the red alga Polysiphonia morrowii has significant antiviral activities against fish pathogenic viruses, infectious hematopoietic necrosis virus (IHNV) and infectious pancreatic necrosis virus (IPNV). The present study was conducted to identify compounds attributed for its antiviral activities and investigate their antiviral activities against IHNV and IPNV. Activity-guided fractionation for 80% methanolic extract of Polysiphonia morrowii using a cell-based assay measuring virus-induced cytopathic effect (CPE) on cells yielded a 90% methanolic fraction, which showed the highest antiviral activity against both viruses among fractions yielded from the extract. From the fraction, two bromophenols were isolated and identified as 3-bromo-4,5-dihydroxybenzyl methyl ether (1) and 3-bromo-4,5-dihydroxybenzaldehyde (2) based on spectroscopic analyses. For both compounds, the concentrations to inhibit 50% of flounder spleen cell (FSP cell) proliferation (CC₅₀) and each viral replication (EC₅₀) were measured. In the pretreatment test, 3-bromo-4,5-dihydroxybenzyl methyl ether (1) and 3-bromo-4,5-dihy-droxybenzaldehyde (2) exhibited significant antiviral activities showing selective index values (SI = CC₅₀/EC₅₀) of 20 to 42 against both IHNV and IPNV. In direct virucidal test, 3-bromo-4,5-dihydroxybenzyl methyl ether (1) showed significant antiviral activités against both viruses while 3-bromo-4,5-dihydroxybenzaldehyde (2) was significantly effective against only IHNV. Although antiviral efficacies of both compounds against IHNV and IPNV were lower than those of ribavirin used as a positive control, our findings suggested that the red alga Polysiphonia morrowii and isolated two bromophenols may have potential as a therapeutic agent against fish viral diseases.     

Natural mutations in IFITM3 modulate post‐translational regulation and toggle antiviral specificity

The interferon‐induced transmembrane (IFITM) proteins protect host cells from diverse virus infections. IFITM proteins also incorporate into HIV‐1 virions and inhibit virus fusion and cell‐to‐cell spread, with IFITM3 showing the greatest potency. Here, we report that amino‐terminal mutants of IFITM3 preventing ubiquitination and endocytosis are more abundantly incorporated into virions and exhibit enhanced inhibition of HIV‐1 fusion. An analysis of primate genomes revealed that IFITM3 is the most ancient antiviral family member of the IFITM locus and has undergone a repeated duplication in independent host lineages. Some IFITM3 genes in nonhuman primates, including those that arose following gene duplication, carry amino‐terminal mutations that modify protein localization and function. This suggests that “runaway” IFITM3 variants could be selected for altered antiviral activity. Furthermore, we show that adaptations in IFITM3 result in a trade‐off in antiviral specificity, as variants exhibiting enhanced activity against HIV‐1 poorly restrict influenza A virus. Overall, we provide the first experimental evidence that diversification of IFITM3 genes may boost the antiviral coverage of host cells and provide selective functional advantages.     

Virucidal activity and the antiviral mechanism of acidic polysaccharides against Enterovirus 71 infection in vitro

Enterovirus 71 (EV71) is the predominant pathogen for severe hand, foot, and mouth disease (HFMD) in children younger than 5 years, and currently no effective drugs are available for EV71. Thus, there is an urgent need to develop new drugs for the control of EV71 infection. In this study, LJ04 was extracted from Laminaria japonica using diethylaminoethyl cellulose-52 with 0.4 mol/l NaCl as the eluent, and its virucidal activity was evaluated based on its cytopathic effects on a microplate. LJ04 is composed of fucose, galactose, and mannose and mainly showed good virucidal activity against EV71. The antiviral mechanisms of LJ04 were the direct inactivation of the virus, the blockage of virus binding, disruptions to viral entry, and weak inhibitory activity against the nonstructural protein 3C. The two most important findings from this study were that LJ04 inhibited EV71 proliferation in HM1900 cells, which are a human microglia cell line, and that LJ04 can directly inactivate EV71 within 2 hr at 37°C. This study demonstrates for the first time the ability of a polysaccharide from L. japonica to inhibit viral and 3C activity; importantly, the inhibition of 3C might have a minor effect on the antiviral effect of LJ04. Consequently, our results identify LJ04 as a potential drug candidate for the control of severe EV71 infection in clinical settings.     

Synthesis,and in vitro Enzymatic and Antiviral Evaluation of d4T Polyphosphate Derivatives as Chain Terminators

A series of d4T di‐ or triphosphate derivatives have been synthesized and evaluated as effective substrates for HIV‐1 RT, and also tested for their in vitro anti‐HIV activity. The steady‐state kinetic study of compounds 1 – 4 in an enzymatic incorporation assay by HIV‐1 RT follows Michaelis? Menten profile. In addition, compounds 2 – 4 are able to inhibit HIV‐1 replication to the same extent as d4T and d4TMP in MT‐4 cells, as well as in CEM/0 cells and CEM/TK^? cells. The data suggests that these d4T polyphosphate derivatives are hydrolyzed to d4T and rephosphorylated to d4TTP before exerting their antiviral activity.     

Optimization of unique, uncharged thioesters as inhibitors of HIV replication

A combinatorial chemistry approach was employed to prepare a restricted library of N-substituted S-acyl-2-mercaptobenzamide thioesters. It was shown that many members of this chemotype display anti-HIV activity via their ability to interact with HIV-1, HIV-2, SIV-infected cells, cell-free virus, and chronically and latently infected cells in a manner consistent with targeting of the highly conserved HIV-1 NCp7 zinc fingers. Compounds were initially screened using two different in vitro antiviral assays and evaluated for stability in neutral buffer containing 10% pooled human serum using a spectrophotometric assay. These data revealed that there was no significant correlation between thioester stability and antiviral activity, however, a slight inverse correlation between serum stability and virucidal activity was noted. Based on the virucidal capability and the ability to select lead compounds to inhibit virus expression from latently infected TNF-induced U1 cells, we next determined if these compounds could prevent HIV cell-to-cell transmission. Several thioesters demonstrated potent inhibition of HIV cell-to-cell transmission with EC₅₀ values in the 80–100 nM range. Thus, we have optimized a series of restricted thioesters and provided evidence that serum stability is not required for antiviral activity. Moreover, selected compounds show potential for development as topical microbicides.     

Antiviral efficacy of Limonium densiflorum against HSV-1 and influenza viruses

Viral infections remain a major threat to humans and animals and there is a crucial need for new antiviral agents especially with the development of resistant viruses. Several Limonium genus members (Plumbaginacea) have been widely used in traditional medicine for the treatment of infections. In this study, we investigated the antiviral activities of different fractions after successive extraction (hexane, dichloromethane, ethanol and methanol) of the halophyte Limonium densiflorum against H1N1 influenza and HSV-1 herpes viruses. In addition, TLC phytochemicals of the shoot extracts were analyzed. All extracts were tested for their cytotoxicity using a fluorometric resazurin assay. The antiviral activity of extracts was tested using four modes of action: virucidal test, pretreatment of cells with samples before infection, attachment assay and plaque reduction test. A good antiviral activity was found with ethanol and methanol extracts. They were most potent in HSV-1 inhibition than H1N1 influenza virus. The most potent inhibition was observed with ethanol extract, and it exhibited high levels of virucidal activity against HSV-1 (IC₅₀ = 6 μg/mL). It inhibits the replication of the virus by 75% when added after penetration of the virus, and by 100% when added during the viral attachment. It protects MDCK cells against influenza virus by abolishing virus to entry into the host cell (IC₅₀ = 55 μg/mL). After attachment of influenza virus, the ethanol extract displayed an appreciable inhibition of virus replication (IC₅₀ = 193 μg/mL). Methanol extract showed a moderate antiviral capacity against both viruses. While dichloromethane has excellent antiherpes potential, results were inappropriate because it was toxic to Vero cells, hexane extract has no effect. TLC analysis of these extracts showed that flavonoids and saponins were the major classes of natural products found in the shoot extracts that may be responsible for these antiviral activities.     

Antiviral activity of crude extracts of Eugenia jambolana Lam. against highly pathogenic avian influenza (H5N1) virus

Crude extracts of leaves and bark of E. jambolana were tested for antiviral activity against highly pathogenic avian influenza virus (H5N1) by CPE reduction assay in three different layouts to elucidate virucidal, post-exposure and preexposure antiviral activity of the extracts. The cold and hot aqueous extracts of bark and hot aqueous extract of leaves of E. jambolana showed significant virucidal activity (100% inhibition) which was further confirmed in virus yield reduction assay (-98 to 99% reduction) and by egg based in ovo assay. The selective index (CC50/EC50) of hot aqueous extract (248) and cold aqueous extract (43.5) of bark of E. jambolana showed their antiviral potential against H5N1 virus. The significant virucidal activity of leaves and bark of E. jambolana merits further investigation as it may provide alternative antiviral agent for managing avian influenza infections in poultry farms and potential avian-human transmission.     

Antiviral activities of flavonoids isolated from the bark of <Emphasis Type="Italic">Rhus verniciflua</Emphasis> stokes against fish pathogenic viruses <Emphasis Type="Italic">In Vitro</Emphasis>

An 80% methanolic extract of Rhus verniciflua Stokes bark showed significant anti-viral activity against fish pathogenic infectious hematopoietic necrosis virus (IHNV) and viral hemorrhagic septicemia virus (VHSV) in a cell-based assay measuring virus-induced cytopathic effect (CPE). Activity-guided fractionation and isolation for the 80% methanolic extract of R. verniciflua yielded the most active ethyl acetate fraction, and methyl gallate (1) and four flavonoids: fustin (2), fisetin (3), butin (4) and sulfuretin (5). Among them, fisetin (3) exhibited high antiviral activities against both IHNV and VHSV showing EC₅₀ values of 27.1 and 33.3 μM with selective indices (SI = CC₅₀/EC₅₀) more than 15, respectively. Fustin (2) and sulfuretin (5) displayed significant antiviral activities showing EC50 values of 91.2–197.3 μM against IHNV and VHSV. In addition, the antiviral activity of fisetin against IHNV and VHSV occurred up to 5 hr post-infection and was not associated with direct virucidal effects in a timed addition study using a plaque reduction assay. These results suggested that the bark of R. verniciflua and isolated flavonoids have significant anti-viral activity against IHNV and VHSV, and also have potential to be used as anti-viral therapeutics against fish viral diseases.     

Chemical composition of Propolis Extract ACF® and activity against herpes simplex virus

Propolis Extract ACF^® (PPE) is a purified extract manufactured from propolis collected in a Canadian region rich in poplar trees, and it is the active substance of a topical ointment used against herpes labialis (cold sores or fever blisters). Aim of this study was to analyze the chemical composition of PPE in order to understand the plant origin and possible relations between compounds and antiviral activity, and to characterize the antiviral activity of the extract against herpes simplex virus in vitro.Material and methodsThe analysis of the propolis extract samples was conducted by Gas Chromatography–Mass Spectrometry (GC–MS). The antiviral activity was tested against herpes simplex viruses type 1 and type 2 in MDBK cell cultures by treating the cells with PPE at the time of virus adsorption, and by incubating the virus with the extract before infection (virucidal assay).ResultsResults from the GC–MS analyses revealed a dual plant origin of PPE, with components derived from resins of two different species of poplar. The chemical composition appeared standardized between extract samples and was also reproduced in the sample of topical ointment. The antiviral studies showed that PPE had a pronounced virucidal effect against herpes simplex viruses type 1 and type 2, and also interfered with virus adsorption.     

PVP-coated silver nanoparticles block the transmission of cell-free and cell-associated HIV-1 in human cervical culture

Background  

Previous in vitro studies have demonstrated that polyvinylpyrrolidone coated silver nanoparticles (PVP-coated AgNPs) have antiviral activity against HIV-1 at non-cytotoxic concentrations. These particles also demonstrate broad spectrum virucidal activity by preventing the interaction of HIV-1 gp120 and cellular CD4, thereby inhibiting fusion or entry of the virus into the host cell. In this study, we evaluated the antiviral activity of PVP-coated AgNPs as a potential topical vaginal microbicide to prevent transmission of HIV-1 infection using human cervical culture, an in vitro model that simulates in vivo conditions.     

Synthesis and Antiviral Evaluation of 3′-Deoxy-β-L-erythro-pentofuranosyl Nucleosides of the Five Naturally Occurring Nucleic Acid Bases

Abstract

The hitherto unknown title compounds were stereospecifically synthesized by glycosylation of pyrimidine and purine aglycons with a suitably peracylated 3′-deoxy-β-L-erythro-pentofuranose, followed by removal of the protecting groups. All the prepared compounds were tested for their ability to inhibit the replication of a variety of DNA and RNA viruses (including HIV), but they did not show significant antiviral activity.     

Ellagitannins from Tuberaria lignosa as entry inhibitors of HIV

Screening of plants from the Iberian Peninsula for anti-human immunodeficiency virus (-HIV) activity revealed that aqueous extract of Tuberaria lignosa gave positive results. Following an activity-guided procedure, the crude extract was counterextracted, and the subsequent fractions obtained tested for their anti-HIV activity in vitro. The bioassay-guided fractionation of the extract afforded an ellagitannin enriched fraction (EEF) isolated for the first time from this species. This EEF exhibited antiviral activity against HIV in MT-2 infected cells, with an IC₅₀ value of 2.33 μg/ml (selectivity index greater than 21). Inhibition of HIV infection by EEF appears to be mediated by CD4 down-regulation, the main receptor for HIV entry. CXCR4 and CCR5 receptors were not affected by EEF, explaining why EEF is able to inhibit R5 and X4 infections.     

Protective Effects of Caffeine on Chikungunya and Zika Virus Infections: An in Vitro and in Silico Study

Infection by viruses Chikungunya (CHIKV) and Zika (ZIKV) continue to be serious problems in tropical and subtropical areas of the world. Here, we evaluated the antiviral and virucidal activity of caffeine against CHIKV and ZIKV in Vero, A549, and Huh-7 cell lines. Results showed that caffeine displays antiviral properties against both viruses. By pre-and post-infection treatment, caffeine significantly inhibited CHIKV and ZIKV replication in a dose-dependent manner. Furthermore, caffeine showed a virucidal effect against ZIKV. Molecular docking suggests the possible binding of caffeine with envelope protein and RNA-dependent RNA polymerase of CHIKV and ZIKV. This is the first study that showed an antiviral effect of caffeine against CHIKV and ZIKV. Although further studies are needed to better understand the mechanism of caffeine-mediated repression of viral replication, caffeine appears to be a promising compound that could be used for in vivo studies, perhaps in synergy with other compounds present in daily beverages.     

Comparison of disc diffusion, Etest and broth microdilution for testing susceptibility of carbapenem-resistant P. aeruginosa to polymyxins

Background

New prophylactic and therapeutic tools are needed for the treatment of herpes simplex virus infections. Several essential oils have shown to possess antiviral activity in vitro against a wide spectrum of viruses.

Aim

The present study was assess to investigate the activities of the essential oil obtained from leaves of Artemisia arborescens against HSV-1 and HSV-2

Methods

The cytotoxicity in Vero cells was evaluated by the MTT reduction method. The IC₅₀ values were determined by plaque reduction assay. In order to characterize the mechanism of action, yield reduction assay, inhibition of plaque development assay, attachment assay, penetration assay and post-attachment virus neutralization assay were also performed.

Results

The IC₅₀ values, determined by plaque reduction assay, were 2.4 and 4.1 μg/ml for HSV-1 and HSV-2, respectively, while the cytotoxicity assay against Vero cells, as determined by the MTT reduction method, showed a CC₅₀ value of 132 μg/ml, indicating a CC₅₀/IC₅₀ ratio of 55 for HSV-1 and 32.2 for HSV-2. The antiviral activity of A. arborescens essential oil is principally due to direct virucidal effects. A poor activity determined by yield reduction assay was observed against HSV-1 at higher concentrations when added to cultures of infected cells. No inhibition was observed by attachment assay, penetration assay and post-attachment virus neutralization assay. Furthermore, inhibition of plaque development assay showed that A. arborescens essential oil inhibits the lateral diffusion of both HSV-1 and HSV-2.

Conclusion

This study demonstrates the antiviral activity of the essential oil in toto obtained from A. arborescens against HSV-1 and HSV-2. The mode of action of the essential oil as antiherpesvirus agent seems to be particularly interesting in consideration of its ability to inactivate the virus and to inhibit the cell-to-cell virus diffusion.     

Carnosine exhibits significant antiviral activity against Dengue and Zika virus

Dengue virus (DENV) and Zika virus (ZIKV) are flaviviruses transmitted to humans by their common vector, Aedes mosquitoes. DENV infection represents one of the most widely spread mosquito‐borne diseases whereas ZIKV infection occasionally re‐emerged in the past causing outbreaks. Although there have been considerable advances in understanding the pathophysiology of these viruses, no effective vaccines or antiviral drugs are currently available. In this study, we evaluated the antiviral activity of carnosine, an endogenous dipeptide (β‐alanyl‐l ‐histidine), against DENV serotype 2 (DENV2) and ZIKV infection in human liver cells (Huh7). Computational studies were performed to predict the potential interactions between carnosine and viral proteins. Biochemical and cell‐based assays were performed to validate the computational results. Mode‐of‐inhibition, plaque reduction, and immunostaining assays were performed to determine the antiviral activity of carnosine. Exogenous carnosine showed minimal cytotoxicity in Huh7 cells and rescued the viability of infected cells with EC₅₀ values of 52.3 and 59.5 μM for DENV2 and ZIKV infection, respectively. Based on the mode‐of‐inhibition assays, carnosine inhibited DENV2 mainly by inhibiting viral genome replication and interfering with virus entry. Carnosine antiviral activity was verified with immunostaining assay where carnosine treatment diminished viral fluorescence signal. In conclusion, carnosine exhibited significant inhibitory effects against DENV2 and ZIKV replication in human liver cells and could be utilized as a lead peptide for the development of effective and safe antiviral agents against DENV and ZIKV.     

The use of bacteriophages of Bacteroides fragilis as indicators of the efficiency of virucidal products

The potential use of bacteriophage B40-8 of Bacteroides fragilis for the evaluation of the virucidal activity of antiseptics or disinfectants was investigated. The antiviral activity of two antiseptics and two disinfectants was evaluated according to a standard guideline. The effect of the virucidal agents was assessed on (i) viruses usually spread by direct contact with surfaces with contaminated secretions, i.e. herpes virus 1 and 2, and vaccinia virus, and (ii) viruses transmitted by the fecal-oral route, i.e. hepatitis A virus, poliovirus, adenovirus and rotavirus. The survival of B40-8 always equalled or exceeded that of the animal viruses tested. Our data suggest the use of bacteriophage B40-8 to complement the information furnished by some standardized methods in ascertaining the antiviral activity of virucidal preparations.     

In vitro anti-influenza virus activities of sulfated polysaccharide fractions from <Emphasis Type="Italic">Gracilaria lemaneiformis</Emphasis>

In this paper, in vitro anti-influenza virus activities of sulfated polysaccharide fractions from Gracilaria lemaneiformis were investigated. Cytotoxicities and antiviral activities of Gracilaria lemaneiformis polysaccharides (PGL), Gracilaria lemaneiformis polysaccharide fraction-1 (GL-1), Gracilaria lemaneiformis polysaccharide fraction-2 (GL-2) and Gracilaria lemaneiformis polysaccharide fraction-3 (GL-3) were studied by the Methyl thiazolyl tetrazolium (MTT) method, and the inhibitory effect against Human influenza virus H1-364 induced cytopathic effect (CPE) on MDCK cells were observed by the CPE method. In addition, the antiviral mechanism of PGL was explored by Plaque forming unit (PFU), MTT and CPE methods. The results showed: i) Cytotoxicities were not significantly revealed, and H1-364 induced CPE was also reduced treated with sulfated polysaccharide fractions from Gracilaria lemaneiformis; ii) Antiviral activities were associated with the mass percentage content of sulfate groups in polysaccharide fractions, which was about 13%, in polysaccharides (PGL and GL-2) both of which exhibited higher antiviral activity; iii) A potential antiviral mechanism to explain these observations is that viral adsorption and replication on host cells were inhibited by sulfated polysaccharides from Gracilaria lemaneiformis. In conclusion, Anti-influenza virus activities of sulfated polysaccharide fractions from Gracilaria lemaneiformis were revealed, and the antiviral activities were associated with content of sulfate groups in polysaccharide fractions.     

Evaluation of multibranched peptides as inhibitors of HIV infection

Summary Synthetic polymeric constructions (SPCs) containing the consensus sequence of the HIV-1 surface envelope gycoprotein gp120 V3 loop (GPGRAF) block the fusion between HIV-1- and HIV-2-infected cells and CD4⁺-uninfected lymphocytes. By testing the activity of a series of SPC analogs in a cell-to-cell fusion assay, we found that the most active construction is an eight-branched SPC with the hexapeptide motif GPGRAF. This compound is also able to inhibit the infection of human lymphocytes and macrophages by unrelated isolates of HIV-1 and HIV-2. This antiviral activity is specific, since no toxicity was observed at the concentrations that inhibit HIV replication and syncytia formation. These data suggest that V3 SPCs may represent a new class of therapeutic anti-HIV agents, able to neutralize a wide range of viral isolates in infected individuals.     

Identification of a Novel HIV-1 Inhibitor Targeting Vif-dependent Degradation of Human APOBEC3G Protein

APOBEC3G (A3G) is a cellular cytidine deaminase that restricts HIV-1 replication by inducing G-to-A hypermutation in viral DNA and by deamination-independent mechanisms. HIV-1 Vif binds to A3G, resulting in its degradation via the 26 S proteasome. Therefore, this interaction represents a potential therapeutic target. To identify compounds that inhibit interaction between A3G and HIV-1 Vif in a high throughput format, we developed a homogeneous time-resolved fluorescence resonance energy transfer assay. A 307,520 compound library from the NIH Molecular Libraries Small Molecule Repository was screened. Secondary screens to evaluate dose-response performance and off-target effects, cell-based assays to identify compounds that attenuate Vif-dependent degradation of A3G, and assays testing antiviral activity in peripheral blood mononuclear cells and T cells were employed. One compound, N.41, showed potent antiviral activity in A3G(+) but not in A3G(−) T cells and had an IC₅₀ as low as 8.4 μm and a TC₅₀ of >100 μm when tested against HIV-1_Ba-L replication in peripheral blood mononuclear cells. N.41 inhibited the Vif-A3G interaction and increased cellular A3G levels and incorporation of A3G into virions, thereby attenuating virus infectivity in a Vif-dependent manner. N.41 activity was also species- and Vif-dependent. Preliminary structure-activity relationship studies suggest that a hydroxyl moiety located at a phenylamino group is critical for N.41 anti-HIV activity and identified N.41 analogs with better potency (IC₅₀ as low as 4.2 μm). These findings identify a new lead compound that attenuates HIV replication by liberating A3G from Vif regulation and increasing its innate antiviral activity.     

Galactan sulfate of Grateloupia indica: Isolation, structural features and antiviral activity

Natural compounds offer interesting pharmacological perspectives for antiviral drug development with regard to broad-spectrum antiviral properties and novel modes of action. In this study, we have analyzed polysaccharide fractions isolated from Grateloupia indica. The crude water extract (GiWE) as well as one fraction (F3) obtained by anion exchange chromatography had potent anti-HSV activity. Their inhibitory concentration 50% (IC₅₀) values (0.12-1.06 μg/ml) were much lower than cytotoxic concentration 50% values (>850 μg/ml). These fractions, which were effective antiviral inhibitors if added only during the adsorption period, had very low anticoagulant activity. Furthermore, they had no direct inactivating effect on virions in a virucidal assay. Chemical, chromatographic and spectroscopic methods showed that the active polysaccharide, which has an apparent molecular mass of 60 kDa and negative specific rotation −16° (c 0.2, H₂O), contains α-(1 → 4)- and α-(1 → 3)-linked galactopyranose residues. Sulfate groups, if present, are located mostly at C-2/6 of (1 → 4)- and C-4/6 of (1 → 3)-linked galactopyranosyl units, and are essential for the anti herpetic activity of this polymer.