Ethanol extract and chromatographic fractions of Tamarindus indica stem bark inhibits Newcastle disease virus replication.

CONTEXT: The plethora of ethnomedicinal applications of Tamarindus indica Linn. (Leguminosae), tamarind, includes treatment of human and livestock ailments; preparations are recognized antipyretics in fevers, laxatives and carminatives. African folklore has various applications of tamarind. However, in Nyasaland, domestic fowl are fed with preparations for prophylactic properties. OBJECTIVES: The objective of this study is to evaluate the antiviral properties of T. indica extract. MATERIALS AND METHODS: Tamarindus indica stem bark was extracted through ethanol maceration over 24 h, and the crude extract was fractionated by gravity-propelled column chromatography. Newcastle disease virus (NDV) inhibitory activity of extract and fractions were evaluated in vivo using 10-d-old embryonated chicken egg (ECE) as the medium for virus cultivation and antivirus assay. About 240 ECE were grouped into eight (three controls and five experimental) and, 200 µL of the extract and fractions respectively inoculated into NDV pre-infected eggs and incubated at 37 °C. Allantoic fluid was harvested 5 d post-virus infection and assayed for haemagglutination (HA). RESULTS: Anti-NDV assessment showed 62.5 mg/mL of crude extract and fractions: TiA, TiC and TiD to yield a HA titre of 1:128 each, while TiB showed 1:64 HA titre. At 125 mg/mL, a titre of 1:16 was recorded against TiB and TiD and, 1:8 against TiA. Similarly, crude extract and TiC, each recorded 1:4 HA titre. However, the minimum concentrations of extract and fraction for virus inactivation were 0.24 mg/mL and 0.49 mg/mL, respectively. CONCLUSION: The antiviral activity shown by T. indica portends novel antiviral drugs and, perhaps, as scaffold for new drugs.

The preventive and therapeutic potential of natural polyphenols on influenza.

Influenza virus belongs to orthomyxoviridae family. This virus is a major public health problems, with high rates of morbidity and mortality. Despite a wide range of pharmacotherapeutic choices inhibiting specific sequences of pathological process of influenza, developing more effective therapeutic options is an immediate challenge. In this paper, a comprehensively review of natural polyphenolic products used worldwide for the management of influenza infection is presented. Cellular and molecular mechanisms of the natural polyphenols on influenza infection including suppressing virus replication cycle, viral hemagglutination, viral adhesion and penetration into the host cells, also intracellular transductional signaling pathways have been discussed in detail. Based on cellular, animal, and human evidence obtained from several studies, the current paper demonstrates that natural polyphenolic compounds possess potential effects on both prevention and treatment of influenza, which can be used as adjuvant therapy with conventional chemical drugs for the management of influenza and its complications.

Qiangzhi decoction protects mice from influenza A pneumonia through inhibition of inflammatory cytokine storm.

OBJECTIVE: To investigate the preventive effects of Qiangzhi Decoction (, QZD) on influenza A pneumonia through inhibition of inflammatory cytokine storm in vivo and in vitro. METHODS: One hundred ICR mice were randomly divided into the virus control, the Tamiflu control and the QZD high-, medium-, and low-dose groups. Mice were infected intranasally with influenza virus (H1N1) at 10 median lethal dose (LD50). QZD and Tamiflu were administered intragastrically twice daily from day 0 to day 7 after infection. The virus control group was treated with distilled water alone under the same condition. The number of surviving mice was recorded daily for 14 days after viral infection. The histological damage and viral replication and the expression of inflammatory cytokines were monitored. Additionally, the suppression capacity on the secretion of regulated on activation normal T cells expressed and secreted (RANTES) and tumor necrosis factor-α (TNF-α) in epithelial and macrophage cell-lines were evaluated. RESULTS: Compared with the virus control group, the survival rate of the QZD groups significantly improved in a dose-dependent manner (P<0.05), the viral titers in lung tissue was inhibited (P<0.05), and the production of inflammatory cytokines interferon-γ (IFN-γ), interleukin-6 (IL-6), TNF-α, and intercellular adhesion molecule-1 (ICAM-1) were suppressed (P<0.05). Meanwhile, the secretion of RANTETS and TNF-α by epithelial and macrophage cell-lines was inhibited with the treatment of QZD respectively in vitro (p<0.05) CONCLUSIONS: The preventive effects of QZD on influenza virus infection might be due to its unique cytokine inhibition mechanism. QZD may have significant therapeutic potential in combination with antiviral drugs.

A polyphenol-rich extract from Chaenomeles sinensis (Chinese quince) inhibits influenza A virus infection by preventing primary transcription in vitro.

ETHNOPHARMACOLOGICAL RELEVANCE: The fruits of Chaenomeles sinensis Koehne (Chinese quince) are distributed throughout China and Japan. It has traditionally been known to have a therapeutic effect against respiratory symptoms caused by infectious diseases. AIM OF THE STUDY: The polyphenol-rich extract, CSD3, from Chaenomeles sinensis has previously been shown to neutralize influenza virus infectivity. The aim of this study was to clarify which step(s) in the replication cycle in vitro were inhibited. MATERIALS AND METHODS: We examined cell-binding, hemagglutination and hemolytic activities and infectivity of A/Udorn/72(H3N2) virus after pre-treatment with CSD3. We also investigated the time course of synthesis for viral mRNA, cRNA, and vRNA in Madin-Darby canine kidney epithelial cells (MDCK) cells infected with CSD3-treated virus. Finally, we studied the effect of CSD3-treatment on the ultrastructure of the influenza virion. RESULTS: Pre-treatment with CSD3 mildly reduced cell-binding, hemagglutination and hemolytic activities. These activities were reduced by 70% to be equivalent to 30% of the control at 1µg/ml. CSD3 severely reduced infectivity to 1% of the control at 1µg/ml. Primary transcription in MDCK cells infected with CSD3 (1µg/ml)-treated virus was decreased to about 1% of that in cells infected with mock-treated virus. Synthesis of viral cRNA, vRNA and secondary mRNA was also severely decreased. Electron microscopy revealed that the integrity of the virus envelope was damaged by CSD3 and was permeable to uranyl acetate. CONCLUSIONS: The main target step(s) of CSD3 in the replication cycle is after cell-binding but before or at primary transcription. Involvement of the increased permeability of virus envelope as the inhibition mechanism was proposed. CSD3 could be useful in preventing influenza virus infection, and be employed as a lozenge or mouthwash for daily use.

Anti-influenza virus activity of Ginkgo biloba leaf extracts.

We examined the influence of Ginkgo biloba leaf extract (EGb) on the infectivity of influenza viruses in Madin-Darby canine kidney (MDCK) cells. Plaque assays demonstrated that multiplication of influenza viruses after adsorption to host cells was not affected in the agarose overlay containing EGb. However, when the viruses were treated with EGb before exposure to cells, their infectivity was markedly reduced. In contrast, the inhibitory effect was not observed when MDCK cells were treated with EGb before infection with influenza viruses. Hemagglutination inhibition assays revealed that EGb interferes with the interaction between influenza viruses and erythrocytes. The inhibitory effect of EGb was observed against influenza A (H1N1 and H3N2) and influenza B viruses. These results suggest that EGb contains an anti-influenza virus substance(s) that directly affects influenza virus particles and disrupts the function of hemagglutinin in adsorption to host cells. In addition to the finding of the anti-influenza virus activity of EGb, our results demonstrated interesting and important insights into the screening system for anti-influenza virus activity. In general, the plaque assay using drug-containing agarose overlays is one of the most reliable methods for detection of antiviral activity. However, our results showed that EGb had no effects either on the number of plaques or on their sizes in the plaque assay. These findings suggest the existence of inhibitory activities against the influenza virus that were overlooked in past studies.

Amelioration of influenza virus-induced reactive oxygen species formation by epigallocatechin gallate derived from green tea.

AIM: To study whether epigallocatechin gallate (EGCG), a green tea-derived polyphenol, exerted anti-influenza A virus activity in vitro and in vivo. METHODS: Madin-Darby canine kidney (MDCK) cells were tested. The antiviral activity of EGCG in the cells was determined using hemagglutination assay and qPCR. Time of addition assay was performed to determine the kinetics of inhibition of influenza A by EGCG. The level of reactive oxygen species (ROS) were determined with confocal microscopy and flow cytometry. BALB/c mice were treated with EGCG (10, 20 or 40 mg·kg(-1)·d(-1), po) for 5 d. On the 3rd d of the treatment, the mice were infected with influenza A virus. Histopathological changes, lung index and virus titers in the lungs were determined. RESULTS: Treatment of influenza A-infected MDCK cells with EGCG (1.25-100 nmol/L) inhibited influenza A replication in a concentration-dependent manner (the ED(50) value was 8.71±1.11 nmol/L). Treatment with EGCG (20 nmol/L) significantly suppressed the increased ROS level in MDCK cells following influenza A infection. In BALB/c mice infected with influenza virus, oral administration of EGCG (40 mg·kg(-1)·d(-1)) dramatically improved the survival rate, decreased the mean virus yields and mitigated viral pneumonia in the lungs, which was equivalent to oral administration of oseltamivir (40 mg·kg(-1)·d(-1)), a positive control drug. CONCLUSION: The results provide a molecular basis for development of EGCG as a novel and safe chemopreventive agent for influenza A infection.

EPs® 7630 (Umckaloabo®), an extract from Pelargonium sidoides roots, exerts anti-influenza virus activity in vitro and in vivo.

A prodelphinidin-rich extract from Pelargonium sidoides DC, EPs® 7630 (Umckaloabo®), which is licensed to treat respiratory tract infections such as acute bronchitis, was investigated for its antiviral effects. EPs® 7630 showed dose-dependent anti-influenza activity at non-toxic concentrations against pandemic H1N1, oseltamivir-sensitive and -resistant seasonal H1N1, seasonal H3N2 and the laboratory H1N1 strain A/Puerto Rico/8/34, while it had no antiviral activity against adenovirus or measles virus. The extract inhibited an early step of influenza infection and impaired viral hemagglutination as well as neuraminidase activity. However, EPs® 7630 did not exhibit a direct virucidal effect, as virus preincubation (unlike cell preincubation) with the extract did not influence infectivity. Importantly, EPs® 7630 showed no propensity to resistance development in vitro. Analysis of EPs® 7630 constituents revealed that prodelphinidins represent the active principle. Chain length influenced antiviral activity, as monomers and dimers were less effective than oligo- and polymers. Importantly, gallocatechin and its stereoisomer epigallocatechin exert antiviral activity also in their monomeric form. In addition, EPs® 7630 administered by inhalation significantly improved survival, body weight and body temperature of influenza-infected mice, without obvious toxicity, demonstrating the benefit of EPs® 7630 in treatment of influenza.

Antiviral effects of Psidium guajava Linn. (guava) tea on the growth of clinical isolated H1N1 viruses: its role in viral hemagglutination and neuraminidase inhibition.

Rapid evolution of influenza RNA virus has resulted in limitation of vaccine effectiveness, increased emergence of drug-resistant viruses and occurrence of pandemics. A new effective antiviral is therefore needed for control of the highly mutative influenza virus. Teas prepared by the infusion method were tested for their anti-influenza activity against clinical influenza A (H1N1) isolates by a 19-h influenza growth inhibition assay with ST6Gal I-expressing MDCK cells (AX4 cells) using fluorogenic quantification and chromogenic visualization. Guava tea markedly inhibited the growth of A/Narita/1/2009 (amantadine-resistant pandemic 2009 strain) at an IC(50) of 0.05% and the growth of A/Yamaguchi/20/06 (sensitive strain) and A/Kitakyushu/10/06 (oseltamivir-resistant strain) at similar IC(50) values ranging from 0.24% to 0.42% in AX4 cells, being 3.4- to 5.4-fold more potent than green tea (IC(50) values: 0.27% for the 2009 pandemic strain and 0.91% to 1.44% for the seasonal strains). In contrast to both teas, oseltamivir carboxylate (OC) demonstrated high potency against the growth of A/Narita/1/09 (IC(50) of 3.83nM) and A/Yamaguchi/20/06 (IC(50) of 11.57nM) but not against that of A/Kitakyushu/10/06 bearing a His274-to-Tyr substitution (IC(50) of 15.97µM). Immunofluorescence analysis under a confocal microscope indicated that both teas inhibited the most susceptible A/Narita/1/2009 virus at the initial stage of virus infection. This is consistent with results of direct inhibition assays showing that both teas inhibited viral hemagglutination at concentrations comparable to their growth inhibition concentrations but inhibited sialidase activity at about 8-times higher concentrations. Guava tea shows promise to be efficacious for control of epidemic and pandemic influenza viruses including oseltamivir-resistant strains, and its broad target blockage makes it less likely to lead to emergence of viral resistance.

Influenza virus variation in susceptibility to inactivation by pomegranate polyphenols is determined by envelope glycoproteins.

Pomegranates have high levels of polyphenols (PPs) and may be a rich source of compounds with antiviral activity. We evaluated the direct anti-influenza activity of three commercially available pomegranate extracts: pomegranate juice (PJ), a concentrated liquid extract (POMxl), and a 93% PP powder extract (POMxp). The acidity of PJ and POMxl solutions contributed to rapid anti-influenza activity, but this was not a factor with POMxp. Studies using POMxp showed that 5min treatment at room temperature with 800µg/ml PPs resulted in at least a 3log reduction in the titers of influenza viruses PR8 (H1N1), X31 (H3N2), and a reassortant H5N1 virus derived from a human isolate. However, the antiviral activity was less against a coronavirus and reassortant H5N1 influenza viruses derived from avian isolates. The loss of influenza infectivity was frequently accompanied by loss of hemagglutinating activity. PP treatment decreased Ab binding to viral surface molecules, suggesting some coating of particles, but this did not always correlate with loss of infectivity. Electron microscopic analysis indicated that viral inactivation by PPs was primarily a consequence of virion structural damage. Our findings demonstrate that the direct anti-influenza activity of pomegranate PPs is substantially modulated by small changes in envelope glycoproteins.

In vitro inhibition of human influenza A virus infection by fruit-juice concentrate of Japanese plum (Prunus mume SIEB. et ZUCC).

Using a plaque reduction assay, treatment of human influenza A viruses with the fruit-juice concentrate of Japanese plum (Prunus mume SIEB. et ZUCC) showed strong in vitro anti-influenza activity against human influenza A viruses before viral adsorption, but not after viral adsorption, with 50% inhibitory concentration (IC50) values against A/PR/8/34 (H1N1) virus, A/Aichi/2/68 (H3N2) virus and A/Memphis/1/71 (H3N2) virus of 6.35+/-0.17, 2.84+/-1.98 and 0.53+/-0.10 microg/ml, respectively. The plum-juice concentrate exhibited hemagglutination activity toward guinea pig erythrocytes. Its hemagglutination activity was inhibited by the monosaccharide N-acetylneuraminic acid and a sialoglycoprotein (fetuin), but not by the other tested monosaccharides (mannose, galactose, glucose and N-acetylglucosamine), suggesting the presence of a lectin-like molecule(s) in the Japanese plum-juice concentrate. Our findings suggest that the fruit-juice concentrate of Japanese plum may prevent and reduce infection with human influenza A virus, possibly via inhibition of viral hemagglutinin attachment to host cell surfaces by its lectin-like activity.

Inhibition of the infectivity of influenza virus by tea polyphenols.

(-)Epigallocatechin gallate (EGCg) and theaflavin digallate (TF3) (1-10 microM) inhibited the infectivity of both influenza A virus and influenza B virus in Madin-Darby canine kidney (MDCK) cells in vitro. Study by electron microscope revealed that EGCg and TF3 (1 mM) agglutinated influenza viruses as well as did antibody, and that they prevented the viruses from adsorbing to MDCK cells. EGCg and TF3 more weakly inhibited adsorption of the viruses to MDCK cells. EGCg and TF3 (1-16 microM) also inhibited haemagglutination by influenza viruses. These findings suggest that tea polyphenols bind to the haemagglutinin of influenza virus, inhibit its adsorption to MDCK cells, and thus block its infectivity.

Effect of electric charged molecules on Sindbis virus hemagglutination and hemolysis.

The role of electrostatic interactions in the attachment and fusion at acidic pH of Sindbis virus (SNV) with goose erythrocytes was studied, investigating the effect of several anionic and cationic polyelectrolytes on SNV hemagglutination and hemolysis. In order to establish the target of active drugs, the compounds were incubated either with the virus particles or with the erythrocytes. Dextran sulfate was the only compound able to inhibit the attachment of SNV to the erythrocytes. Fusion of virus with red cells was reduced dose-dependently by the polyanions dextran sulfate, mucin and polygalacturonic acid. On the contrary two polycations, polylysine and polybrene, enhanced viral hemolytic activity. However the effect of polyions is not exclusively related to the electric charge since ineffective molecules were found in both classes of compounds.

Inhibition of influenza virus haemolytic and haemagglutination activities by monoclonal antibodies to haemagglutinin glycopolypeptides HA1 and HA2.

Acid treatment of influenza virus enhanced haemagglutination inhibiting (HI) activity of some anti-HA1 monoclonal antibodies (MoAbs). These changes in the HI-activity could be either due to alteration in the mutual orientation of MoAb (e.g. IC8, IB8) binding epitope to receptor site or to an increase in the number of epitopes accessible to the corresponding MoAbs (e.g. IVA1). HI test with pH 5-virus revealed similar (although not identical) antigenic differences among related virus strains as the HI test with pH 7-virus. Anti-HA2 MoAbs were negative in the HI test with both pH 5- and pH 7-virus. Anti-HA1 MoAbs showed a HI activity with pH 5-treated BHA similar to that with pH 5-treated virus. Surprisingly one out of eight anti-HA2 MoAbs (IIF4) exhibited a relatively high HI activity to pH 5-BHA-mediated haemagglutination. Virus-induced red blood cell haemolysis was efficiently inhibited with several anti-HA1 MoAbs (e.g. IC8, IB8, and IIB4) while other anti-HA1 antibodies, including IVA1 and IVG6 with preferential reactivity with pH 5-treated antigens in RIA, gave no inhibition. As a rule, anti-HA2 MoAbs were poor haemolysis inhibitors.

Ultrastructure and some biological properties of influenza A virus. I. Alterations in the activity of surface antigens of influenza A virus by enzymes and infectious activity of such changed viruses.

Strain of A/ZSRR/053/74 (H3N2) virus was subjected to the action of enzymes. Treatment of the virus with soluble trypsin and trypsin bound to a carrier for different periods of time resulted in decreased neuraminidase activity and infectivity, however, hemagglutinin activity was preserved. After treatment pickled with soluble bromelain, virions of decreased hemagglutinin amount but preserved neuraminidase activity, were obtained. This was accompanied by a slight fall in the infectivity. Bromeline bound to a carrier produced different results. It exerted a destructive effect on neuraminidase activity, decreasing virus infectivity and only slightly hemagglutinin titer. Chromatographically purified fractions of virus with very low hemagglutinin titer and undeterminable neuraminidase, did not completely loose their infectivity.

[Mechanism of action of 2-(1'-aminoethyl)-bicyclo[2.2.1]heptane hydrochloride]. / O mekhanizme deistviia 2-(1'-aminoétil)-bitsiklo[2.2.1]geptan khlorgidrata.

The effect of 2-[1'-aminoethyl)-bicyclo[2.2.1]heptane chlorohydrate on the accumulation of hemagglutinating activity of chick embryo fibroblasts infected with influenza A/FPV/Rostock/34 (Hav1N1) and A/WSN/33 (H0N1) was studied. The drug inhibited hemagglutinines accumulation when added at various intervals after infection, the maximum effect having been observed upon its addition to the maintenance medium immediately after adsorption. Polyacrylamide gel electrophoresis showed the drug to reduce the synthesis of virus-specific proteins of A/FPV and to inhibit considerably the transcriptase activity of A/FPV and A/WSN viruses in vitro.