Susceptibility of betanodavirus in a newly established vertebra-derived cell line from Mosquitofish (Gambusia affinis).

In the present study, a new cell line from the vertebra of mosquitofish Gambusia affinis was successfully established and characterized. The cell line is named as bone Gambusia affinis (BGA) and subcultured for more than 55 passages in Leibovitz's/L15 medium supplemented with 15% FBS at 28°C. The cell line has a modal chromosome number of 48. Molecular characterization of the partial sequence of the coi gene confirmed the origin of the BGA cell line from mosquitofish. These cells exhibited epithelial morphology confirmed by the cytokeratin marker. The BGA cells showed mineralization of their extracellular matrix when stained with alizarin red and von Kossa stain. BGA cells were found to be susceptible to RGNNV and SJNNV strains of betanodavirus (NNV) showing cytopathic effect with multiple vacuolations in the cells. The RT-PCR confirmed the betanodavirus infections in BGA cells. The SEM micrograph showed the morphological changes observed in the cell during virus infection. The in vivo challenge experiment also showed the viral replicating efficiency in the Gambusia affinis with increasing viral titre. Thus, our present results show that the BGA cell line is a useful tool for isolating betanodavirus and could be used to investigate bone cell differentiation and extracellular matrix mineralization.

Antiviral activity of pinocembrin against Zika virus replication.

Zika virus (ZIKV) is a mosquito-borne virus that has garnered a lot of attention in recent years, due to the explosive epidemic from 2014 to 2016. Since its introduction in the Americas in late 2014, ZIKV has spread at an unprecedented rate and scale throughout the world and infected millions of people. Its infection has also been associated with severe neurological disorders like Guillain-Barré syndrome and microcephaly in fetuses. Despite these, there is currently no approved antiviral against ZIKV. In this study, an immunofluorescence-based high throughput screen was conducted on a library of 483 flavonoid derivatives to identify potential anti-ZIKV compounds. Flavonoids, which are natural polyphenolic compounds found in plants, represent an attractive source of antivirals due to their abundance in food and expected low toxicity. From the primary screen, three hits were selected for validation by cell viability and viral plaque reduction assays. Pinocembrin, a flavanone found in honey, tea and red wine, was chosen for downstream studies as it exhibited the strongest inhibition of ZIKV infection in human placental JEG-3 cells (IC50 = 17.4 µM). Time-course studies revealed that pinocembrin acts on post-entry process(es) of the ZIKV replication cycle. Furthermore, pinocembrin inhibits viral RNA production and envelope protein synthesis based on quantitative reverse transcription-PCR (qRT-PCR) and Western blot analyses. This study has demonstrated for the first time the in vitro anti-ZIKV activity of pinocembrin.

Phenylalanine and Phenylglycine Analogues as Arginine Mimetics in Dengue Protease Inhibitors.

Dengue virus is an increasingly global pathogen. One of the promising targets for antiviral drug discovery against dengue and related flaviviruses such as West Nile virus is the viral serine protease NS2B-NS3. We here report the synthesis and in vitro characterization of potent peptidic inhibitors of dengue virus protease that incorporate phenylalanine and phenylglycine derivatives as arginine-mimicking groups with modulated basicity. The most promising compounds were (4-amidino)-L-phenylalanine-containing inhibitors, which reached nanomolar affinities against dengue virus protease. The type and position of the substituents on the phenylglycine and phenylalanine side chains has a significant effect on the inhibitory activity against dengue virus protease and selectivity against other proteases. In addition, the non-natural, basic amino acids described here may have relevance for the development of other peptidic and peptidomimetic drugs such as inhibitors of the blood clotting cascade.

Design, synthesis and evaluation of novel anti-HCV molecules that deliver intracellularly three highly potent NS5A inhibitors.

The design and synthesis of new non-symmetrical NS5A inhibitors with sulfur containing amino acids is reported along with their ability to block HCV replication in an HCV 1b replicon system. These compounds display EC50 values in the picomolar range with a large therapeutic index (>10(6)). Moreover, cellular pharmacology studies show that our preferred compounds intracellularly deliver three potent NS5A inhibitors.

Synthesis and Biological Evaluation of a Series of 2-((1-substituted-1H-1,2,3-triazol-4-yl)methylthio)-6-(naphthalen-1-ylmethyl)pyrimidin-4(3H)-one As Potential HIV-1 Inhibitors.

A series of novel S-DABO derivatives with the substituted 1,2,3-triazole moiety on the C-2 side chain were synthesized using the simple and efficient CuAAC reaction, and biologically evaluated as inhibitors of HIV-1. Among them, the most active HIV-1 inhibitor was compound 4-((4-((4-(2,6-dichlorobenzyl)-5-methyl-6-oxo-1,6-dihydropyrimidin-2-ylthio)methyl)-1H-1,2,3-triazol-1-yl)methyl)benzenesulfonamide (B5b7), which exhibited similar HIV-1 inhibitory potency (EC50  = 3.22 µm) compared with 3TC (EC50  = 2.24 µm). None of these compounds demonstrated inhibition against HIV-2 replication. The preliminary structure-activity relationship (SAR) of these new derivatives was discussed briefly.

Oligolysine-conjugated zinc(II) phthalocyanines as efficient photosensitizers for antimicrobial photodynamic therapy.

A series of zinc(II) phthalocyanines conjugated with an oligolysine chain (n=2, 4, and 8) were synthesized and characterized by using various spectroscopic methods. As shown by using UV/Vis and fluorescence spectroscopic methods, these compounds were nonaggregated in N,N-dimethylformamide, and gave a weak fluorescence emission and high singlet oxygen quantum yield (Φ(Δ) =0.86-0.89) as a result of their di-α-substitution. They became slightly aggregated in water with 0.05 % Cremophor EL, but they could still generate singlet oxygen effectively. The antimicrobial photodynamic activities of these compounds were then examined against various bacterial strains, including the Gram-positive methicillin-sensitive Staphylococcus aureus ATCC 25923 and methicillin-resistant Staphylococcus aureus ATCC BAA-43, and the Gram-negative Escherichia coli ATCC 35218 and Pseudomonas aeruginosa ATCC 27853. Generally, the dyes were much more potent toward the Gram-positive bacteria. Only 15 to 90 nM of these photosensitizers was required to induce a 4 log reduction in the cell viability of the strains. For Escherichia coli, the photocytotoxicity increased with the length of the oligolysine chain. The octalysine derivative showed the highest potency with a 4 log reduction concentration of 0.8 µM. Pseudomonas aeruginosa was most resistant to the photodynamic treatment. The potency of the tetralysine derivative toward a series of clinical strains of Staphylococcus aureus was also examined and found to be comparable with that toward the nonclinical counterparts. Moreover, the efficacy of these compounds in photodynamic inactivation of viruses was also examined. They were highly photocytotoxic against the enveloped viruses influenza A virus (H1N1) and herpes simplex virus type 1 (HSV1), but exhibited no significant cytotoxicity against the nonenveloped viruses adenovirus type 3 (Ad3) or coxsackievirus (Cox B1). The octalysine derivative also showed the highest potency with an IC(50) value of 0.05 nM for the two enveloped viruses.

Antiviral activity of polymethoxylated flavones from "Guangchenpi", the edible and medicinal pericarps of citrus reticulata 'Chachi'.

The present study found that the supercritical fluid extract of "Guangchenpi" possessed in vitro antiviral activity against respiratory syncytial virus (RSV). Bioassay-guided isolation and identification of this extract led to obtain five active polymethoxylated flavones (1-5). Cytopathic effect (CPE) reduction assay exhibited that tangeretin (2) and nobiletin (3), two major polymethoxylated flavones in the extract, possessed better anti-RSV effect comparable to the positive control ribavirin. Plaque reduction assay revealed that tangeretin dose-dependently inhibited RSV-induced plaque formation on the HEp-2 cells. This polymethoxylated flavone mainly affected the intracellular replication of RSV, and it also could inhibit RSV entry into the HEp-2 cells. Further investigations with quantitative real-time PCR and confocal and Western blot assays indicated that tangeretin downregulated the expression of RSV phosphoprotein (P protein). Results suggest the potential application of the supercritical fluid extract of "Guangchenpi" and tangeretin in the treatment and the prevention of RSV infection.

H3N2 homeopathic influenza virus solution modifies cellular and biochemical aspects of MDCK and J774G8 cell lines.

BACKGROUND: Influenza viruses cause highly contagious acute respiratory illnesses with significant mortality, especially among young children, elderly people, and individuals with serious medical conditions. This encourages the development of new treatments for human flu. Biotherapies are diluted solutions prepared from biological products compounded following homeopathic procedures. OBJECTIVES: To develop a biotherapy prepared from the infectious influenza A virus (A/Aichi/2/68 H3N2) and to verify its in vitro response. METHODS: The ultradiluted influenza virus solution was prepared in the homeopathic dilution 30dH, it was termed Influenzinum RC. The cellular alterations induced by this preparation were analyzed by optical and electron microscopy, MTT and neutral red assays. Glycolytic metabolism (PFK-1) was studied by spectrophotometric assay. Additionally, the production of tumor necrosis factor-α (TNF-α) by J774.G8 macrophage cells was quantified by ELISA before and after infection with H3N2 influenza virus and treatment. RESULTS: Influenzinum RC did not cause cytotoxic effects but induced morphological alterations in Madin-Darby canine kidney (MDCK) cells. After 30 days, a significant increase (p < 0.05) in mitosis rate was detected compared to control. MDCK mitochondrial activity was changed after treatment for 10 and 30 days. Treatment significantly diminished (p < 0.05) PFK-1 activity. TNF-α in biotherapy-stimulated J774.G8 macrophages indicated a significant (p < 0.05) increase in this cytokine when the cell supernatant was analyzed. CONCLUSION: Influenzinum RC altered cellular and biochemical features of MDCK and J774G8 cells.

A new conjugated amide-dimer from the aerial parts of Piper submultinerve.

Bioassay-guided fractionation and purification of the aerial parts of Piper submultinerve led to the isolation of a new conjugated amide-dimer, submultinamide A (1), along with 11 known compounds. The structures were determined on the basis of spectroscopic methods. Among the tested compounds, pellitorine (2), guineensine (4), N-benzylcinnamide (6) and aristolactam BII (8) showed significant activities in the anti-syncytium assay using (ΔTat/Rev)MC99 virus and 1A2 cell line system, whereas 2 was most active (EC50 35.1 µM and selectivity index 4.7). In the HIV-1 reverse transcriptase assay, only 4 was active with IC50 50.8 µM.

Establishment of a stable cell line coexpressing dengue virus-2 and green fluorescent protein for screening of antiviral compounds.

This study aimed to generate a stable cell line harboring subgenomic dengue virus replicon and a green fluorescent gene (DENV/GFP) for a cell-based model to screen anti-DENV compounds. The gene-encoding envelope protein of DENV-2 was deleted and then replaced with fragments of the GFP gene and a foot-and-mouth-disease virus 2A-derived cleavage site. The human cytomegalovirus immediate early and antisense hepatitis delta virus ribozyme sequences were added at the 5'- and 3'-ends. An internal ribosome entry site and neomycin resistance genes were placed upstream and next to the NS1 gene. The recombinant plasmids were propagated in a mammalian cell line. A stable cell line with the brightest green fluorescent protein and the highest viral protein and RNA expression was selected from six clones. The clone was then examined for effectiveness in an antiviral drug screening assay with compounds isolated from the local plants using two known antiviral agents as controls. Two novel flavones, PMF and TMF, were discovered having DENV-inhibitory properties. The data were validated by a conventional plaque titration assay. The results indicate that this newly developed cell line is efficient for use as a cell-based model for primary screening of anti-DENV compounds.

Clonal vaccinia virus grown in cell culture as a new smallpox vaccine.

Although the smallpox virus was eradicated over 20 years ago, its potential release through bioterrorism has generated renewed interest in vaccination. To develop a modern smallpox vaccine, we have adapted vaccinia virus that was derived from the existing Dryvax vaccine for growth in a human diploid cell line. We characterized six cloned and one uncloned vaccine candidates. One clone, designated ACAM1000, was chosen for development based on its comparability to Dryvax when tested in mice, rabbits and monkeys for virulence and immunogenicity. By most measures, ACAM1000 was less virulent than Dryvax. We compared ACAM1000 and Dryvax in a randomized, double-blind human clinical study. The vaccines were equivalent in their ability to produce major cutaneous reactions ('takes') and to induce neutralizing antibody and cell-mediated immunity against vaccinia virus.

Inhibitors of the IMPDH enzyme as potential anti-bovine viral diarrhoea virus agents.

Ribavirin and mycophenolic acid (MPA) are known inhibitors of the IMPDH enzyme (E.C. 1.1.1.205). This enzyme catalyzes the conversion of inosine monophosphate to xanthine monophosphate, leading eventually to a decrease in the intracellular level of GTP and dGTP. The antiviral effect against bovine viral diarrhoea virus (BVDV) of 15 analogues related to MPA was determined. MDBK cells were infected with the cytopathic strain of BVDV in presence or absence of test compounds. Viral RNA was extracted from the cell supernatant fluids and quantified by RT-PCR. Ribavirin showed a potent antiviral effect against BVDV with 90% effective concentration (EC90) of 4 microM. MPA along with several analogues, including both its corresponding aldehyde and alcohol, and modifications in the length of the side chain (C2- and C4-derivatives) were tested. We have identified previously unreported IMPDH inhibitors that have potent anti-BVDV activity, namely: C6-MPAlc (5), C6-MPA-Me (7), C4-MPAlc (8), C4-MPA (10) and C2-MAD (20). Most of these compounds inhibited the IMPDH enzyme in the nanomolar range (4-800 nM) in cell-free assays. Some compounds, such as mizoribine, which is a potent inhibitor of IMPDH in vitro (enzyme 50% inhibitory concentration IC50=4 nM), had no detectable anti-BVDV activity up to 100 microM. The compounds were essentially non-toxic to a confluent monolayer of MDBK cells. However, in exponentially growing cells, they showed minimal toxicity at 100 microM over a 24 h period, but the toxicity was more pronounced after 3 days [50% cytotoxic concentration (CC50) value ranged from 5 to 30 microM].

Inhibition of the replication of a hepatitis C virus-like RNA template by interferon and 3'-deoxycytidine.

The development of low molecular weight inhibitors of hepatitis C virus (HCV) replication has been hindered by the lack of a good cell-based system that models the entire HCV replication cycle. To date the only two therapies approved for the treatment of HCV infection are interferon (IFN)-alpha and the nucleoside analogue, ribavirin. We have created a cell-based system that allows for the accurate quantification of the replication of an HCV-like RNA template by proteins that are encoded for by the HCV genome. The system consists of a cell line that constitutively produces luciferase in response to the production of functional HCV replicative proteins. The 293B4alpha cell line has been formatted into a semi-high throughput, cell-based screen for inhibitors of HCV replication. When these cells were treated with either IFN-alpha or -beta, luciferase production decreased in a dose-responsive manner. Counterscreening these molecules in another cell line, 293SVLuc, in which luciferase production in not dependent the presence of functional HCV proteins, showed that the inhibition of luciferase in the 293B4alpha cell line was due to inhibition of the replication of the HCV-like RNA template and not anti-cellular or -luciferase activity. Moreover, when the 293B4alpha cell line was treated with the ribonucleoside analogue, 3'-deoxycytidine, luciferase decreased in a dose-responsive manner. 3'-deoxyguanosine and 3'-deoxyuridine did not inhibit luciferase production and 3'-deoxyadenosine was too cytotoxic to determine if it had any anti-HCV activity.

Synthesis and anti-HIV activity of prodrugs derived from saquinavir and indinavir.

With a view to improving the pharmacological properties, safety and pharmacokinetic profiles of current protease inhibitors, the synthesis of various acyl-substituted saquinavir and indinavir prodrugs, their in vitro stability with respect to hydrolysis and their anti-HIV (LAI and HTLV IIIB) activity and cytotoxicity in CEM-SS and MT4 cells have been investigated. Hydrolysis of the ester bond and liberation of the active free drug was found to be crucial for HIV inhibition: the faster the hydrolysis, the closer the anti-HIV activity was to that of the respective parent drug. This is the case for most of the C-14-substituted indinavir and saquinavir derivatives (IC50 from 10 to 360 nM for ester half-lives of 90 min to 40 h). Concomitantly, the level of HIV inhibition is very low for the prodrugs for which hydrolysis is very slow. This is the case with the myristoyl or oleyl saquinavir esters, owing to the stable masking of the hydroxyl that is part of the peptidomimetic non-cleavable transition state isostere responsible for the inhibitory potency of saquinavir (and indinavir). In contrast, the anti-HIV activity of the monosubstituted C-8 indinavir prodrugs seems not to be correlated with their resistance to hydrolysis, as expected (the C-8 hydroxyl of indinavir is not involved in the transition state isostere). No cytotoxicity was detected for the indinavir and saquinavir prodrugs for concentrations as high as 10 or even 100 microM, thus indicating promising therapeutic potential.

Extract of Prunella vulgaris spikes inhibits HIV replication at reverse transcription in vitro and can be absorbed from intestine in vivo.

It has been reported that extracts of the spike of Prunella vulgaris (PS) exhibit anti-HIV activity at the adsorption and reverse transcription stages. In this study, the actual activity of PS in cells, kinetic analysis of the inhibitory activity of PS against HIV reverse transcriptase and the feasibility of oral administration were examined. First, to clarify whether this extract shows anti-HIV activity in cells in vitro, the number of copies of proviral DNA in HIV-exposed cells was calculated. The number of copies was significantly decreased in cells cultured in the presence of PS extract, but not in the presence of dextran sulphate. The activity of PS extract in the cells was also assessed by the drug addition test, during and after HIV adsorption. PS extract and dextran sulphate suppressed HIV production to similar levels when added after HIV adsorption. However, only PS extract suppressed HIV production at the same concentration when the drugs were added during HIV adsorption. Presumably, the penetration of the PS extract into the cells was required for this activity. Secondly, fractionated PS inhibited HIV reverse transcription in a non-competitive manner. This fractionated PS kept anti-HIV activity, but inhibited HIV replication and adsorption to a lesser extent compared to dextran sulphate. Lastly, an active component(s) was detected in plasma in vivo, after injection into the intestine, which demonstrates the feasibility of oral administration dosing.

Production of influenza virus in serum-free mammalian cell cultures.

Human influenza viruses are routinely isolated and grown in a variety of mammalian cell substrates. However, influenza viruses for use as inactivated vaccine are still produced in embryonated eggs. Using a perfusion culture-based bioreactor process using serum-free medium, both human and equine influenza viruses of different types and subtypes could be produced to high titres. Classical DEAE-dextran microcarriers were found to be more suitable than polyester sponge carriers for virus production. In addition, MDCK cells grown in serum-free medium were further validated as the most suitable cell substrate compared to Vero and BHK-21 C13 cells for large scale virus production of influenza virus. Finally, to minimize potential contamination by adventitious agents, it was demonstrated that a new serum-free medium in which all animal-derived products are replaced by a plant extract, efficiently supports the growth of MDCK cells as well as the production of influenza virus in the presence of trypsin when using the perfusion bioreactor process.

Protective effect of Astragali Radix by intraperitoneal injection against Japanese encephalitis virus infection in mice.

We examined the protective effect of Astragali Radix extracts (AE) by intraperitoneal injection against Japanese encephalitis virus (JEV) infection in mice. A protective effect was observed by all four samples of AE used. However, the degree of effectiveness for each AE was different. The observed survival rates of the groups injected with sample A (from Shanhsi, Japanese name Sansei-syo) and sample D (from Hokkaido) extracts were higher than 80% at 21 d after JEV inoculation. The groups injected with sample B (from Hopei, Japanese name Kahoku-syo) and sample C (from Hsiahsi, Japanese name Sensei-syo) extracts had a 60% survival rate. The increase in hemagglutination inhibition antibody titer was negligible in mice that survived 21 d after JEV inoculation. The antiviral effect of AE was examined by plaque assay in vitro, but no antiviral effect was shown. In mice injected with AE, the peritoneal exudate cell (PEC) numbers increased significantly, compared to the control. In these PEC, active oxygen production was also high. Also the group as a whole displayed a high survival rate against JEV infection, these were so strong. From these results, we propose that the protective effect of AE is dependent on a non-specific mechanism during the early stage of infection, before it shifts to antibody production, and that PEC plays an important role.

The reverse transcriptase assay: problems and practical solutions.

The detection of retroviruses has become critical for addressing the safety concerns associated with biologically derived products, including those derived from blood and cell line substrates, and gene therapy based systems. Most, if not all, retroviruses encode a unique enzyme called reverse transcriptase whose RNA-dependent DNA polymerase activity can be used as a marker for detecting retroviral contamination in test material. In this presentation we document some practical concerns when using the reverse transcriptase assay for detection of retroviruses. We also illustrate important aspects in the assay design by presentation of results that needed supplemental testing to enable accurate assessment of retroviral contamination.

In vitro antiviral activities of sulfated polysaccharides from a marine microalga (Cochlodinium polykrikoides) against human immunodeficiency virus and other enveloped viruses.

A marine microalga, Cochlodinium polykrikoides, produces extracellular sulfated polysaccharides. Isolation and purification of the polysaccharides were accomplished by precipitation with ethanol and Cetavlon, followed by DEAE-cellulose column chromatography (polysaccharides A1 and A2). These polysaccharides, which were homogeneous when analysed by both ultracentrifugal and electrophoretic methods, were composed of mannose, galactose, glucose and uronic acid, together with sulfate groups (S = 7-8% w/w). Both A1 and A2 inhibited the cytopathic effect of influenza virus types A and B in MDCK cells, that of respiratory syncytial virus types A and B in HEp-2 cells, that of human immunodeficiency virus type 1 in MT-4 cells; and, except A1 for herpes simplex virus type 1 and A2 for parainfluenza virus type 2 in HMV-2 cells, the cochlodinium polysaccharides showed no antiviral activity against parainfluenza virus types 2 and 3, measles virus, mumps virus or herpes simplex virus type 1 in HMV-2 cells. No cytotoxicity for host cells was observed with these polysaccharides at a concentration of 100 micrograms ml-1. Inhibitory effects on various viruses were achieved at concentrations that were not markedly inhibitory to the blood coagulation process.