Identification and characterization of new potent inhibitors of dengue virus NS5 proteinase from Andrographis paniculata supercritical extracts on in animal cell culture and in silico approaches.

ETHNOPHARMACOLOGICAL RELEVANCE: About 2.5 billion peoples are at risk of dengue virus and the majority of people, use traditional plant-based medicines to combat dengue. The whole plant of Andrographis paniculata used traditionally over past decades for health promotion. Andrographolide isolated from Andrographis paniculata is used as natural remedy for the treatment of various diseases in different parts of the world. Andrographolide has been reported to have antiviral activity against hepatitis B virus, hepatitis C virus, herpes simplex virus, influenza virus, chikungunya virus, dengue virus 2 and 4. AIM OF THE STUDY: The aim of the present study to isolate the andrographolide from the A. paniculata by supercritical fluid extraction technique and to characterize the isolated compound along with it anti-dengue activity against DENV-2 in vitro and in silico methods. MATERIALS AND METHODS: Supercritical extraction condition for A. paniculata was standardised to isolate andrographolide compound at definite temperature and pressure on the basis of previous study. The andrographolide was identified by using Ultraviolet-Visible Spectroscopy (UV-VIS), Fourier-Transform Infrared Spectroscopy (FT-IR) and High Performance Thin Layer Chromatography (HPTLC) and Proton Nuclear Magnetic Resonance (1HNMR). The maximum non-toxic dose of isolated andrographolide was detected by MTT assay using a micro plate reader at 595 nm. One hundred (100) copies/ml of the DENV-2 virus was used for antiviral assay in C6/36 cells lines and inhibition of virus due to andrographolide was determined by real-time PCR assay. The purity of isolated andrographolide was determined by Differential Scanning Calorimetry (DSC). The dengue NS5 receptor protein was docked with andrographolide and evaluated on the basis of the total energy and binding affinity score by Auto Dock (V4.2.6) software. RESULTS: Andrographolide, a diterpene lactone was isolated from the A. paniculata supercritical extract at 40 °C temperature and 15 Mpa pressure. UV spectrophotometer analysis revealed that the curve of andrographolide plant extract was overlapped with reference compound at 228 nm and the similar bands were detected from FT-IR spectroscopy analysis at 3315, 2917, 2849, 1673, 1462 and 1454 cm-1 in isolated and standard andrographolide. HPTLC analysis shows the retention factor (Rf) of A. paniculata extract at 0.74 ± 0.06 as similar to standard andrographolide Rf values. The purity of isolated andrographolide was 99.76%. The maximum non-toxic dose of isolated andrographolide was found as 15.62 µg/ml on the C6/36 cell line calculated by using MTT assay. The andrographolide showed the 97.23% anti-dengue activity against the dengue-2 virus in C6/36 cell lines. Results of molecular docking showed that the interaction between andrographolide and NS5 of dengue protein with the maximum binding energy as -7.35 kcal/mol. CONCLUSIONS: It is concluded that isolated andrographolide from the A. paniculata possess anti-dengue activity against dengue-2 virus as revealed from in vitro and in silico method. Due to lack of the vaccine and anti-viral agents, andrographolide extracted from A. paniculata play a major role to inhibit the dengue replication. Hence, it could be a source for drug design and help to reduce the dengue infection.

The Geraniin-Rich Extract from Reunion Island Endemic Medicinal Plant Phyllanthus phillyreifolius Inhibits Zika and Dengue Virus Infection at Non-Toxic Effect Doses in Zebrafish.

The mosquito-borne viruses dengue (DENV) and Zika (ZIKV) viruses are two medically important pathogens in tropical and subtropical regions of the world. There is an urgent need of therapeutics against DENV and ZIKV, and medicinal plants are considered as a promising source of antiviral bioactive metabolites. In the present study, we evaluated the ability of Phyllanthus phillyreifolius, an endemic medicinal plant from Reunion Island, to prevent DENV and ZIKV infection in human cells. At non-cytotoxic concentration in vitro, incubation of infected A549 cells with a P. phillyreifolius extract or its major active phytochemical geraniin resulted in a dramatic reduction of virus progeny production for ZIKV as well as four serotypes of DENV. Virological assays showed that P. phillyreifolius extract-mediated virus inhibition relates to a blockade in internalization of virus particles into the host cell. Infectivity studies on ZIKV showed that both P. phillyreifolius and geraniin cause a loss of infectivity of the viral particles. Using a zebrafish model, we demonstrated that administration of P. phillyreifolius and geraniin has no effect on zebrafish locomotor activity while no morbidity nor mortality was observed up to 5 days post-inoculation. Thus, P. phillyreifolius could act as an important source of plant metabolite geraniin which is a promising antiviral compound in the fight against DENV and ZIKV.

Anti-viral activity of culinary and medicinal mushroom extracts against dengue virus serotype 2: an in-vitro study.

BACKGROUND: Dengue is a mosquito-borne viral infection that has become a major public health concern worldwide. Presently, there is no specific vaccine or treatment available for dengue viral infection. METHODS: Lignosus rhinocerotis, Pleurotus giganteus, Hericium erinaceus, Schizophyllum commune and Ganoderma lucidium were selected for evaluation of their in-vitro anti-dengue virus serotype 2 (DENV-2) activities. Hot aqueous extracts (HAEs), ethanol extracts (EEs), hexane soluble extracts (HSEs), ethyl acetate soluble extracts (ESEs) and aqueous soluble extracts (ASEs) were prepared from the selected mushrooms. The cytotoxic effects of the extracts were evaluated by the MTT assay. The anti-DENV-2 activities of the extracts were evaluated in three different assays: simultaneous, attachment and penetration assays were perfomed using plaque reduction assays and RT-qPCR assays. The effect of the addition time on viral replication was assessed by the time of addition assay, and a virucidal assay was carried out to evaluate the direct effect of each mushroom extract on DENV-2. The chemical composition of glucans, and the protein and phenolic acid contents in the extracts were estimated. RESULTS: We found that the HAEs and ASEs of L. rhinocerotis, P. giganteus, H. erinaceus and S. commune were the least toxic to Vero cells and showed very prominent anti-DENV2 activity. The 50% inhibitory concentration (IC50) values of the ASEs ranged between 399.2-637.9 µg/ml, while for the HAEs the range was 312.9-680.6 µg/ml during simultaneous treatment. Significant anti-dengue activity was also detected in the penetration assay of ASEs (IC50: 226.3-315.4 µg/ml) and HAEs (IC50: 943.1-2080.2 µg/ml). Similarly, we observed a marked reduction in the expression levels of the ENV and NS5 genes in the simultaneous and penetration assays of the ASEs and HAEs. Time-of-addition experiments showed that the highest percent of anti-DENV2 activity was observed when the mushroom extracts were added immediately after virus adsorption. None of the extracts exhibited virucidal effect. Chemical composition analysis showed that the major components in the mushroom HAEs and ASEs were glucan (beta D-glucan) and proteins, however, there was no significant correlation between the anti-dengue activity and the concentration of glucans and proteins. CONCLUSION: These findings demonstrated the potential of mushroom extracts as anti-dengue therapeutic agents with less toxic effects.

In-silico identification and evaluation of plant flavonoids as dengue NS2B/NS3 protease inhibitors using molecular docking and simulation approach.

Dengue infection is prevailing among the people not only from the developing countries but also from the developed countries due to its high morbidity rate around the globe. Hence, due to the unavailability of any suitable vaccine for rigorous dengue virus (DENV), the only mode of its treatment is prevention. The circumstances require an urgent development of efficient and practical treatment to deal with these serotypes. The severe effects and cost of synthetic vaccines simulated researchers to find anti-viral agents from medicinal plants. Flavonoids present in medicinal plants, holds anti-viral activity and can be used as vaccine against viruses. Therefore, present study was planned to find anti-viral potential of 2500 flavonoids inhibitors against the DENVNS2B/NS3 protease through computational screening which can hinder the viral replication within the host cell. By using molecular docking, it was revealed that flavonoids showed strong and stable bonding in the binding pocket of DENV NS2B/NS3 protease and had strong interactions with catalytic triad. Drug capability and anti-dengue potential of the flavonoids was also evaluated by using different bioinformatics tools. Some flavonoids effectively blocked the catalytic triad of DENV NS2B/NS3 protease and also passed through drug ability evaluation. It can be concluded from this study that these flavonoids could act as potential inhibitors to stop the replication of DENV and there is a need to study the action of these molecules in-vitro to confirm their action and other properties.

Garlic Organosulfur Compounds Reduce Inflammation and Oxidative Stress during Dengue Virus Infection.

Dengue virus (DENV) is a mosquito-borne flavivirus that causes significant global human disease and mortality. One approach to develop treatments for DENV infection and the prevention of severe disease is through investigation of natural medicines. Inflammation plays both beneficial and harmful roles during DENV infection. Studies have proposed that the oxidative stress response may be one mechanism responsible for triggering inflammation during DENV infection. Thus, blocking the oxidative stress response could reduce inflammation and the development of severe disease. Garlic has been shown to both reduce inflammation and affect the oxidative stress response. Here, we show that the garlic active compounds diallyl disulfide (DADS), diallyl sulfide (DAS) and alliin reduced inflammation during DENV infection and show that this reduction is due to the effects on the oxidative stress response. These results suggest that garlic could be used as an alternative treatment for DENV infection and for the prevention of severe disease development.

[Antiviral activity in vitro and pharmacokinetics of HCV entry inhibitor AVR560].

Several novel compounds were found to be potent inhibitors of the HCV (JFH-1 isolate) infection in vitro. Human serum did not significantly reduce antiviral activity of the lead compound, AVR560 (< 4-fold). The immunohistochemistry studies with the Huh7 cell line, infectable with the HCV (JFH-1 strain), demonstrated that AVR560 inhibited the early steps of viral infection and blocked the spread of the HCV infection in tissue culture. The cytotoxicity in Huh7 and Vero-76 cell lines was mild. AVR560 proved to be a specific HCV inhibitor and exhibited no activity against other flaviviruses such as yellow fever (strain 17D), West Nile (strain NY99), and dengue (New Guinea type 2) in in vitro infection experiments. AVR560 also did not inhibit any of the tested human CYP450 isozymes (3A4, 1A2, 2C19 and 2D6). In the pharmacokinetic studies in mice, rats and dogs, favorable pharmacokinetic profiles and good oral bioavailability were observed for AV560. Further pre-clinical studies with this novel HCV inhibitor are in progress.

Compound A, a dissociated glucocorticoid receptor modulator, reduces dengue virus-induced cytokine secretion and dengue virus production.

Dengue Virus (DENV) infection is an important mosquito-borne viral disease and its clinical symptoms range from a predominantly febrile disease, dengue fever (DF), to dengue hemorrhagic fever (DHF) and dengue shock syndrome (DSS). Increased levels of cytokines - the so-called 'cytokine storm', contribute to the pathogenesis of DHF/DSS. In this study, we compared the expression of cytokine genes between mock-infected and DENV-infected HepG2 cells using a real-time PCR array and revealed several up-regulated chemokines and cytokines, including CXCL10 and TNF-α. Compound A (CpdA), a plant-derived phenyl aziridine precursor containing anti-inflammatory action and acting as a dissociated nonsteroidal glucocorticoid receptor modulator, was selected as a candidate agent to modulate secretion of DENV-induced cytokines. CpdA is not a glucocorticoid but has an anti-inflammatory effect with no metabolic side effects as steroidal ligands. CpdA significantly reduced DENV-induced CXCL10 and TNF-α secretion and decreased leukocyte migration indicating for the first time the therapeutic potential of CpdA in decreasing massive immune activation during DENV infection.

Inhibition of dengue virus by targeting viral NS4B protein.

We report a novel inhibitor that selectively suppresses dengue virus (DENV) by targeting viral NS4B protein. The inhibitor was identified by screening a 1.8-million-compound library using a luciferase replicon of DENV serotype 2 (DENV-2). The compound specifically inhibits all four serotypes of DENV (50% effective concentration [EC(50)], 1 to 4 µM; and 50% cytotoxic concentration [CC(50)], >40 µM), but it does not inhibit closely related flaviviruses (West Nile virus and yellow fever virus) or nonflaviviruses (Western equine encephalomyelitis virus, Chikungunya virus, and vesicular stomatitis virus). A mode-of-action study suggested that the compound inhibits viral RNA synthesis. Replicons resistant to the inhibitor were selected in cell culture. Sequencing of the resistant replicons revealed two mutations (P104L and A119T) in the viral NS4B protein. Genetic analysis, using DENV-2 replicon and recombinant viruses, demonstrated that each of the two NS4B mutations alone confers partial resistance and double mutations confer additive resistance to the inhibitor in mammalian cells. In addition, we found that a replication defect caused by a lethal NS4B mutation could be partially rescued through trans complementation. The ability to complement NS4B in trans affected drug sensitivity when a single cell was coinfected with drug-sensitive and drug-resistant viruses. Mechanistically, NS4B was previously shown to interact with the viral NS3 helicase domain; one of the two NS4B mutations recovered in our resistance analysis-P104L-abolished the NS3-NS4B interaction (I. Umareddy, A. Chao, A. Sampath, F. Gu, and S. G. Vasudevan, J. Gen. Virol. 87:2605-2614, 2006). Collectively, the results suggest that the identified inhibitor targets the DENV NS4B protein, leading to a defect in viral RNA synthesis.

Virucidal activity of Colombian Lippia essential oils on dengue virus replication in vitro.

The inhibitory effect of Lippia alba and Lippia citriodora essential oils on dengue virus serotypes replication in vitro was investigated. The cytotoxicity (CC50) was evaluated by the MTT assay and the mode of viral inhibitory effect was investigated with a plaque reduction assay. The virus was treated with the essential oil for 2 h at 37 masculineC before cell adsorption and experiments were conducted to evaluate inhibition of untreated-virus replication in the presence of oil. Antiviral activity was defined as the concentration of essential oil that caused 50% reduction of the virus plaque number (IC50). L. alba oil resulted in less cytotoxicity than L. citriodora oil (CC50: 139.5 vs. 57.6 microg/mL). Virus plaque reduction for all four dengue serotypes was observed by treatment of the virus before adsorption on cell. The IC50 values for L. alba oil were between 0.4-32.6 microg/mL and between 1.9-33.7 microg/mL for L. citriodora oil. No viral inhibitory effect was observed by addition of the essential oil after virus adsorption. The inhibitory effect of the essential oil seems to cause direct virus inactivation before adsorption on host cell.

Virucidal activity of Colombian Lippia essential oils on dengue virus replication in vitro

The inhibitory effect of Lippia alba and Lippia citriodora essential oils on dengue virus serotypes replication in vitro was investigated. The cytotoxicity (CC50) was evaluated by the MTT assay and the mode of viral inhibitory effect was investigated with a plaque reduction assay. The virus was treated with the essential oil for 2 h at 37ºC before cell adsorption and experiments were conducted to evaluate inhibition of untreated-virus replication in the presence of oil. Antiviral activity was defined as the concentration of essential oil that caused 50 percent reduction of the virus plaque number (IC50). L. alba oil resulted in less cytotoxicity than L. citriodora oil (CC50: 139.5 vs. 57.6 μg/mL). Virus plaque reduction for all four dengue serotypes was observed by treatment of the virus before adsorption on cell. The IC50 values for L. alba oil were between 0.4-32.6 μg/mL and between 1.9-33.7 μg/mL for L. citriodora oil. No viral inhibitory effect was observed by addition of the essential oil after virus adsorption. The inhibitory effect of the essential oil seems to cause direct virus inactivation before adsorption on host cell.

Effect of Hippophae rhamnoides leaf extract against Dengue virus infection in human blood-derived macrophages.

Dengue virus occurs as four distinct serotypes, called Dengue 1, 2, 3, and 4. Symptomatic dengue virus infection ranges from a self limited febrile illness, dengue fever (DF), to a more severe disease, dengue hemorrhagic fever/dengue shock syndrome (DHF/DSS). The anti-Dengue treatment is severely hampered as no specific therapeutic agents are available. Even present treatment strategies for Dengue are more supportive than curative. In the present study anti-dengue activity of Hippophae rhamnoides (Seabuckthorn, SBT) leaf extract was evaluated in Dengue virus type-2 infected blood-derived human macrophages as macrophages are the primary target of Dengue virus infection. Infected cells were treated with SBT leaf extract and compared with commercially available anti-viral drug, Ribavirin. The extract was able to maintain the cell viability of Dengue-infected cells at par with Ribavirin along with the decrease and increase in TNF-alpha and IFN-gamma respectively. Anti-dengue activity of SBT extract was further determined by the traditional plaque assay. These observations suggest that the SBT leaf extract has a significant anti-dengue activity and has the potential for the treatment of Dengue.

Folk knowledge about dengue mosquitoes and contributions of health belief model in dengue control promotion in Northeast Thailand.

The health belief model (HBM) has been adopted as the principal theory for health education and communication for dengue haemorrhagic fever (DHF) prevention and control in Thailand. The HBM focuses on persuading people to acknowledge their vulnerability and susceptibility to DHF, and the benefits of undertaking dengue larval control in household water containers. This study was undertaken in Khon Kaen province to investigate folk knowledge and beliefs about dengue mosquitoes and larval control campaigns and relating these to the theoretical components of HBM. Findings from this study indicate that health education messages can raise awareness during an outbreak but do not ensure sustained larval control practices. Several barriers are identified, including insufficient control agents, inadequate knowledge of control methods, and incompatibility of control practices with people's beliefs. The barriers prevail over the benefits of recommended larval control practices. In developing health education messages, consideration should go beyond the HBM and focus on control methods that are compatible with the socio-cultural environment in which control practices are being encouraged.

In vitro and in vivo antiviral properties of sulfated galactomannans against yellow fever virus (BeH111 strain) and dengue 1 virus (Hawaii strain).

Two galactomannans, one extracted from seeds of Mimosa scabrella, having a mannose to galactose ratio of 1.1, and another with a 1.4 ratio from seeds of Leucaena leucocephala, were sulfated. The products from M. scabrella (BRS) and L. leucocephala (LLS) had a degree of sulfation of 0.62 and 0.50, and an average molecular weight of 620x10(3) and 574x10(3) gmol(-1), respectively. Their activities against yellow fever virus (YFV; BeH111 strain) and dengue 1 virus (DEN-1; Hawaii strain) were evaluated. This was carried out in young mice following intraperitoneal infection with YFV. At a dose of 49 mgkg(-1), BRS and LLS gave protection against death in 87.7 and 96.5% of the mice, respectively. When challenged with 37.5 LD50 of YFV, mice previously inoculated with BRS+virus or LLS+virus, showed 93.3 and 100% resistance, respectively, with neutralization titers similar to mice injected with 25 LD50 of formaldehyde-inactivated YFV. In vitro experiments with YFV and DEN-1 in C6/36 cell culture assays in 24-well microplates showed that concentrations that produced a 100-fold decrease in virus titer of YFV were 586 and 385 mgl(-1) for BRS and LLS, respectively. For DEN-1 they were 347 and 37 mgl(-1), respectively. Sulfated galactomannans, thus demonstrate in vitro and in vivo activity against flaviviruses.

Fetal rhesus monkey lung cells can be grown in serum-free medium for the replication of dengue-2 vaccine virus.

Serum-free media were developed to grow diploid fetal rhesus monkey lung (DBS-FRhL-2) cells and to propagate dengue-type 2 virus vaccine strain PR-159 (dengue-2 vaccine virus). Vitamins, amino acids, growth factors, hormones and other organic compounds, and inorganic salts were substituted for fetal bovine serum. The composition of the medium that was optimal for growth of DBS-FRhL-2 cells differed from medium optimal for the propagation of dengue-2 vaccine virus. Insulin, epidermal growth factor, fibroblast growth factor, and platelet-derived growth factor were required for DBS-FRhL-2 cell proliferation in serum-free medium but were inhibitory for virus propagation. Adenosine, cytidine, guanosine, uridine, and thymidine, each at 0.01 mM concentration, were necessary as medium supplements to obtain a high yield of dengue-2 vaccine virus in DBS-FRhL-2 cells under serum-free conditions. DBS-FRhL-2 cells grown in serum-free medium produced dengue-2 vaccine virus with yields similar to those of cells grown in the presence of serum. Dengue-2 vaccine virus obtained under serum-free conditions retained its phenotypic markers such as temperature sensitivity and small plaque size.

A lipid inhibitor of dengue virus in human colostrum and milk; with a note on the absence of anti-dengue secretory antibody.

Neutralizing activity against dengue virus types 1--4 was observed in milk samples from 5 non-immune and 29 dengue immune women. Anti-dengue activity in milk and colostrum was found only in the lipid component. The inhibitory activity is directed against the virus and not cell surfaces. When immunoglobulin types IgM,IgA, IgG were isolated from colostrum from dengue immune women, no antibody activity was found. Anti-dengue activity in human milk did not decrease over a period of ten months after delivery.