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In Vitro Evaluation of Synergistic Inhibitory Effects of Neuraminidase Inhibitors and Methylglyoxal Against Influenza Virus Infection
Abstract
Influenza virus infections are serious public health concerns worldwide that cause considerable mortality and morbidity. Moreover, the emergence of resistance to anti-influenza viral agents underscores the need to develop new anti-influenza viral agents and novel treatment strategies. Recently, we identified anti-influenza viral activity of manuka honey. Therefore, we hypothesized that methylglyoxal (MGO), a key component of manuka honey, may impart anti-influenza viral activity. The aim of this study was to evaluate the anti-influenza viral activity of MGO and its potential in combination treatments with neuraminidase (NA) inhibitors.
MDCK cells were used to evaluate anti-influenza viral activity. To evaluate the mechanism of MGO, plaque inhibition assays were performed. The synergistic effects of MGO and viral NA inhibitors were tested.
MGO inhibited influenza virus A/WSN/33 replication 50% inhibitory concentration = 240 ± 190 μmol; 50% cytotoxic concentration = 1.4 ± 0.4 mmol; selective index (SI) = 5.8, which is related to its virucidal effects. Moreover, we found that MGO showed promising activity against various influenza strains. A synergistic effect was observed by a marked increase in SI of NA inhibitors at ∼1/100(th) of their single usage. A synergistic effect of MGO and oseltamivir was also observed against oseltamivir-resistant virus.
Our results showed that MGO has potent inhibitory activity against influenza viruses and also enhanced the effect of NA inhibitors. Thus, the co-administration of MGO and NA inhibitors should be considered for treatment of influenza virus infections.
Copyright © 2014 IMSS. Published by Elsevier Inc. All rights reserved.
... We performed screening to select anti-influenza compounds based on CV assays ( Supplementary Fig. S1) because CV staining is an alternative and rapid method for evaluating cytotoxicity and antiviral activities 28,29 . To confirm the correlations between the CV assays and antiviral activities, we compared the antiviral activity of PA-49 based on cell morphology, water-soluble tetrazolium salt (WST-1), CV staining and 50% tissue culture infective dose (TCID 50 ) assays (Fig. 4). ...
... viruses were tested using the CV assay ( Table 2). The EC 50 value of oseltamivir against A/WSN/33 was found to be similar to that reported previously 29 . When PA-49 was used in the analysis, the EC 50 values against A/Virginia/ Tables 1 and 2). ...
... EC 50 WST-1 assay. The WST-1 assay was performed as previously described 29 with some modifications. Briefly, MDCK cells were seeded in 24-well plates at a density of 1.8 × 10 5 cells/well in MEM containing 5% FBS and incubated overnight. ...
Influenza virus infections are serious public health concerns throughout the world. The development of compounds with novel mechanisms of action is urgently required due to the emergence of viruses with resistance to the currently-approved anti-influenza viral drugs. We performed in silico screening using a structure-based drug discovery algorithm called Nagasaki University Docking Engine (NUDE), which is optimised for a GPU-based supercomputer (DEstination for Gpu Intensive MAchine; DEGIMA), by targeting influenza viral PA protein. The compounds selected by NUDE were tested for anti-influenza virus activity using a cell-based assay. The most potent compound, designated as PA-49, is a medium-sized quinolinone derivative bearing a tetrazole moiety, and it inhibited the replication of influenza virus A/WSN/33 at a half maximal inhibitory concentration of 0.47 μM. PA-49 has the ability to bind PA and its anti-influenza activity was promising against various influenza strains, including a clinical isolate of A(H1N1)pdm09 and type B viruses. The docking simulation suggested that PA-49 interrupts the PA-PB1 interface where important amino acids are mostly conserved in the virus strains tested, suggesting the strain independent utility. Because our NUDE/DEGIMA system is rapid and efficient, it may help effective drug discovery against the influenza virus and other emerging viruses.
... The mechanism of MGO is thought to involve direct interaction of MGO on the virus surface and interference with the interactions between viruses and host cells. Because the mode of action of MGO is different from that of NAIs, MGO in combination with NAI could enhance the NAI activity for inhibition of influenza A virus replication (21). ...
... In this study, we compared the anti-influenza viral activity of MGO against influenza B viruses with that of other NAIs and evaluated its potential as a new universal antiviral agent against influenza viruses. In addition, we also compared the susceptibilities to NAIs of several strains of influenza B virus, including laboratory strains and clinically isolated samples from patients in Japan, with those of influenza A viruses reported previously (21). MGO exhibited a broad spectrum of inhibitory activity against influenza B viruses, not only against NAI-sensitive influenza virus B strains, but also against NAI-resistant influenza B strains. ...
... We first evaluated the cytotoxicity of MGO using MDCK cells and determined a CC 50 value of 1.4 ± 0.4 mM. Our study reported that MGO obviously suppressed influenza A virus replication in a strain-independent manner (21). However, the antiviral activity of MGO against influenza B types has not yet been evaluated. ...
Influenza A and B virus infections are serious public health concerns globally. However, the concerns regarding influenza B infection have been underestimated. The currently used anti-influenza drugs have not provided equal efficacy for both influenza A and B viruses. Susceptibility to neuraminidase (NA) inhibitors has been observed to be lower for influenza B viruses than for influenza A viruses. Moreover, the emergence of resistance to anti-influenza drugs underscores the need to develop new drugs. Recently, we reported that methylglyoxal (MGO) suppressed influenza A virus replication in a strain-independent manner. Therefore, we hypothesize that MGO exhibits anti-influenza activity against B strains. This study aimed to evaluate the anti-influenza viral activity of MGO against influenza B strains by using Madin-Darby canine kidney (MDCK) cells. Several types of influenza B viruses were used to determine the activity of MGO. The susceptibilities of influenza A and B viruses to NA inhibitors were compared. MGO inhibited influenza B virus replication, with 50% inhibitory concentrations ranging from 23-140 μM, which indicated greater sensitivity of influenza B viruses than influenza A viruses. Our results show that MGO has potent inhibitory activity against influenza B viruses, including NA inhibitor-resistant strains.
... The water-soluble tetrazolium salt (WST-1) assay was used to evaluate the viability of cells infected with virus in the presence of NUD-1 or oseltamivir as described previously [45], with some modifications. Briefly, MDCK cells were infected for determination of the IC 50 as described above. ...
... Oseltamivir (Fig 4B) showed similar results to NUD-1 (Fig 4A). Cell viability, as determined by a WST-1 assay, was equivalent to that determined by crystal violet staining as previously reported [45]. These data demonstrated that NUD-1 and oseltamivir inhibited virus replication in a similar manner. ...
Influenza viruses have acquired resistance to approved neuraminidase-targeting drugs, increasing the need for new drug targets for the development of novel anti-influenza drugs. Nucleoprotein (NP) is an attractive target since it has an indispensable role in virus replication and its amino acid sequence is well conserved. In this study, we aimed to identify new inhibitors of the NP using a structure-based drug discovery algorithm, named Nagasaki University Docking Engine (NUDE), which has been established especially for the Destination for GPU Intensive Machine (DEGIMA) supercomputer. The hit compounds that showed high binding scores during in silico screening were subsequently evaluated for anti-influenza virus effects using a cell-based assay. A 4-hydroxyquinolinone compound, designated as NUD-1, was found to inhibit the replication of influenza virus in cultured cells. Analysis of binding between NUD-1 and NP using surface plasmon resonance assay and fragment molecular orbital calculations confirmed that NUD-1 binds to NP and could interfere with NP-NP interactions essential for virus replication. Time-of-addition experiments showed that the compound inhibited the mid-stage of infection, corresponding to assembly of the NP and other viral proteins. Moreover, NUD-1 was also effective against various types of influenza A viruses including a clinical isolate of A(H1N1)pdm09 influenza with a 50% inhibitory concentration range of 1.8–2.1 μM. Our data demonstrate that the combined use of NUDE system followed by the cell-based assay is useful to obtain lead compounds for the development of novel anti-influenza drugs.
... Soba honey, kanro honey, acacia honey and renge honey followed manuka honey for effectiveness. Charyasriwong et al. [46] found that methylglyoxal ( Figure 12) was an active ingredient in manuka honey with EC50 values of 180-420 μM against H1N1, H3N2, H5N2 and even oseltamivir-resistant H1N1. They even found that manuka honey had a synergistic effect when combined with NA inhibitors like oseltamivir. ...
... MG has been reported to exhibit antiviral property against footand-mouth disease virus [31], and Newcastle disease virus [32] via interaction with viral RNA. MG exhibited antiviral activity against multiple influenza virus strains (including H1N1, H3N2, H5N2, and oseltamivir-resistant H1N1) [33], in addition to its synergistic effect when administered as a co-treatment with neuraminidase inhibitors. ...
... According to Charyasriwong et al. [72] , hydrogen peroxide, phenols, and bioflavonoids are the major classes of bioactive compounds responsible for antiviral activity against viral infections. Flavonoids are major constituents of honey and play an important role in this activity. ...
Background
Bee bread can be defined as a popular bee product (behind honey, propolis, beewax, bee pollen, royal jelly and apitoxin), a natural substance, a mixture of bee pollen, honey and lactic acid bacteria that is the result of lactic acid bacteria (LAB) fermentation of bee pollen collected by bees from the natural environment, and is the protein basis of food in the beehive.
Results
Primarily, honey bees use bee bread for brood growing (nutrition), but humans use bee bread in folk medicine and apitherapy, but also as a functional food, according to the bread's powerful healing properties and content of different molecules (bioactive, antibacterial, antifungal, antiviral, etc.). The biological properties of bee bread depend on its chemical composition, the melliferous plants in the region, geographical area, season, climatic conditions, etc.
Conclusion
According to its chemical and nutritional properties, bee bread is more valuable (higher biological value, faster digestibility and better chemical composition if compared to bee pollen) than bee pollen. Bee bread contains about 250 different substances, such as proteins, macro- and microelements, lipids, free amino acids, fatty acids (linoleic, linolenic and arachidonic), flavonoids, phenolic compounds, vitamins, and enzymes. Many different scientific articles have been published in international journals with focus on the chemical composition of honey, bee pollen and propolis. This paper contributes to the knowledge of the chemical composition, nutritional value and bioactivity of bee bread.
... Kilty et al. (2011) demonstrated in vitro the effectiveness of Manuka honey against biofilms of methicillin-resistant S. aureus along with thoseof Pseudomonas sp. MGO shows also an antiviral activity against influenza (flu) like treatment with neuraminidase inhibitors(Charyasriwong et al. 2015).Defensin-1 or royalisin. Defensin-1 is one of the four antimicrobial peptides (apidaecin, abaecin, hymenoptaecin, and defensin) secreted by the bee(Ilyasov et al. 2012). ...
Honey is a complex and variable mixture that contains more than 180 biochemical compounds from various molecule families. This mixture is achieved after processing the nectar out of plant food sources at the level of the bees’ abdomen. The bioactive components found in this natural product are in charge of its antimicrobial properties. Honey is used for its antibacterial actions over Gram+ and Gram-; their anti-fungal and antimycotic actions against melds and yeasts, along with its protozoal and antiviral activities. This literature review outlines its naturally antimicrobial potential of honey; explains the factors responsible for this potential; and spell out their mechanisms of action. Osmotic pressure, water activity, acid content of honey, presence of bioactive compounds like: hydrogen peroxide, phenolic acids, flavonoids, the MGO, defensin-1, lysozyme, volatile compounds as well as antibacterial products secreted from the lactic bacteria that are behind this antimicrobial activity. This potential basically depends on the biological activities of the initially harvested floral source, its geographical origin, the season, the storage conditions, the honey age, the health of bees’ colonies and the suitable beekeeping practices.
... Another in vitro study also showed the effectiveness of commercial manuka honey against a HSV-1 isolate using Vero cells (69). Charyasriwong et al. (70) reported that an active ingredient, methylglyoxal, present in manuka honey showed an activity against H3N2, H1N1, H5N2, and also oseltamivir-resistant H1N1. Similarly, berries are rich in bioactive compounds, particularly polyphenolics, flavonoids along with polysaccharides, carotenoids, organic acids, anthocyanins, etc. have been used as a natural cure against upper respiratory tract infections. ...
Nowadays, despite enormous scientific advances, viral diseases remain the leading cause of morbidity worldwide, and their potential to spread is escalating, eventually turning into pandemics. Nutrition can play a major role in supporting the immune system of the body and for the optimal functioning of the cells of the immune system. A healthy diet encompassing vitamins, multi-nutrient supplements, functional foods, nutraceuticals, and probiotics can play a pivotal role in combating several viral invasions in addition to strengthening the immune system. This review provides comprehensive information on diet-based scientific recommendations, evidence, and worldwide case studies in light of the current pandemic and also with a particular focus on virus-induced respiratory tract infections. After reviewing the immune potential of nutraceuticals based on the lab studies and on human studies, it was concluded that bioactive compounds such as nutraceuticals, vitamins, and functional foods (honey, berries, etc.) with proven antiviral efficacy, in addition to pharmaceutical medication or alone as dietary supplements, can prove instrumental in treating a range of virus-induced infections in addition to strengthening the immune system. Milk proteins and peptides can also act as adjuvants for the design of more potent novel antiviral drugs.
... The bacterial urease enzyme produces ammonia, which allows the bacteria to adapt to an acidic environment. Methylglioxal (MGO) was also evaluated for antiviral activity against influenza and its potential as treatment combined with neuraminidase (NA) inhibitors (Charyasriwong et al., 2015). The results showed that the combined administration of MGO and NA inhibitors should be considered for the treatment of influenza virus infections. ...
Honey is a natural product with a sweet flavor. Honey is made by the honeybee (Apis mellifera L.) from the nectar of flowers or other plant secretions that are collected near the hive. These products are mixed with bee saliva and stored. Several studies have demonstrated that honey exhibits antioxidant, antimicrobial, nematicidal, antifungal, anticancer, and anti‐inflammatory activities. These properties are influenced by the plants from which the secretions are harvested, from the naturally occurring compounds present in the nectar. Studies of the properties and applications of honey have distinguished honey from other natural products due to the presence of certain compounds and due its bioactive properties. The focus of this review is to discuss the identified and isolated compounds from monofloral honey produced by A. mellifera, with specific emphasis on antioxidant and antimicrobial properties of honey and its therapeutic health benefits.
... Hydrogen peroxide, phenols and bioflavonoids, found honey bee products are the major classes of bioactive compounds responsible for their antiviral activity against various viral infections (Charyasriwong et al., 2015). Flavonoids are major constituents of honey and play an important role for this activity. ...
Coronavirus Disease (COVID-19) has infected people in 210 nations and has been declared a pandemic on March 12, 2020 by the World Health Organization (WHO). In the absence of effective treatment and/or vaccines for COVID-19, natural products of known therapeutic and antiviral activity could offer an inexpensive, effective option for managing the disease. Benefits of products of honey bees such as honey, propolis, and bee venom, against various types of diseases have been observed. Honey bees products are well known for their nutritional and medicinal values, they have been employed for ages for various therapeutic purposes. In this review, promising effects of various bee products against the emerging pandemic COVID-19 are discussed. Products of honey bees that contain mixtures of potentially active chemicals, possess unique properties that might help to protect, fight, and alleviate symptoms of COVID-19 infection.
... The anti-RSV activities were evaluated by a cytopathic effects (CPE) assay based on previous reports [23][24][25]. In brief, HEp-2 (human epidermoid carcinoma of larynx) cells were seeded in 96-well plates at a density of 2.5 × 10 4 cells/well in 100 µl of Eagle's minimum essential medium (E-MEM) supplemented with 10% fetal bovine serum (FBS), and the cells were then incubated at 37 • C in an atmosphere of 5% CO 2 overnight. ...
On our quest for new bioactive molecules from marine sources, two cyclic imines (1, 2) were isolated from a dinoflagellate extract, inhibiting the growth of the respiratory syncytial virus (RSV). Compound 1 was identified as a known molecule portimine, while 2 was elucidated to be a new cyclic imine, named kabirimine. The absolute stereochemistry of 1 was determined by crystallographic work and chiral derivatization, whereas the structure of 2 was elucidated by means of spectroscopic analysis and computational study on all the possible isomers. Compound 1 showed potent cytotoxicity (CC50 < 0.097 µM) against HEp2 cells, while 2 exhibited moderate antiviral activity against RSV with IC50 = 4.20 µM (95% CI 3.31–5.33).
... Methylglyoxal is reported to show antiviral activity against certain strains of in uenza virus (Charyasriwong et al. 2015) and foot and mouth disease virus (Ghizatullina 1976). Methylglyoxal bis-(guanylhydrazone) inhibits vaccinia virus proliferation by inhibiting S-adenosyl-L-methionine decarboxylase in HeLa cells (Williamson 1976). ...
Of the many dietary factors associated with inflammation and oxidative stress, a specific group are food-derived pro-inflammatory and pro-oxidant compounds, so-called advanced glycation end products (AGEs). While AGEs have been recognized as factors in the pathogenesis of diabetic complications, the importance of AGEs of dietary origin as a factor in human disease is of more recent concern. This book presents data from the past two decades on the role of AGEs in causing chronic disease. It starts by defining the compounds passing through all the clinical diseases that have been associated with them and finishes by offering different therapeutic options to deal with the problem.
... To date, some extracts and food constituents have demonstrated antiviral effects: Vigna angularis (red bean) extract against rabies virus, 4 Litchi chinensis (Lychee fruit) extract against betanodavirus, 5 and whey acidic proteins against human immunodeficiency virus (HIV). 6 In addition, extracts from Alchemilla mollis (Lady's mantle tea), 7 Aspalathus linearis (Rooibos tea), 8 Panax ginseng (Red ginseng), 9 purified constituents such as methylglyoxal from manuka honey, 10,11 lactoferrin from bovine milk, 12 catechins from green tea, 13 and a dihydrochalcone from seagrass Thalassodendron ciliatum (Forsk.) den Hartog 14 have been shown to possess potent anti-influenza virus activity. ...
The high propensity of influenza viruses to develop resistance to antiviral drugs necessitates the continuing search for new therapeutics. Peanut skins, which are low-value byproducts of the peanut industry, are known to contain high levels of polyphenols. In this study, we investigated the antiviral activity of ethanol extracts of peanut skins against various influenza viruses using cell-based assays. Extracts with a higher polyphenol content exhibited higher antiviral activities, suggesting that the active components are the polyphenols. An extract prepared from roasted peanut skins effectively inhibited the replication of influenza virus A/WSN/33 with a half maximal inhibitory concentration of 1.3 μg/mL. Plaque assay results suggested that the extract inhibits the early replication stages of the influenza virus. It demonstrated activity against both influenza type A and type B viruses. Notably, the extract exhibited a potent activity against a clinical isolate of the 2009 H1N1 pandemic, which had reduced sensitivity to oseltamivir. Moreover, a combination of peanut skin extract with the anti-influenza drugs, oseltamivir and amantadine, synergistically increased their antiviral activity. These data demonstrate the potential application of peanut skin extract in the development of new therapeutic options for influenza management.
... Indeed, previous reports have stated advantages of synthetic NAI combinations over single-drug influenza treatment [21][22][23][24]. Additionally, drug-herb combinations such as the Manuka honey constituent methylglyoxal along with oseltamivir [25] as well as also some entirely herbal-based combinations [26] proved to be effective anti-influenza NAIs. ...
Neuraminidase is a key enzyme in the life cycle of influenza viruses and is present in some bacterial pathogens. We here assess the inhibitory potency of plant tannins versus clinically used inhibitors on both a viral and a bacterial model neuraminidase by applying the 2′-(4-methylumbelliferyl)-α-d-N-acetylneuraminic acid (MUNANA)-based activity assay. A range of flavan-3-ols, ellagitannins and chemically defined proanthocyanidin fractions was evaluated in comparison to oseltamivir carboxylate and zanamivir for their inhibitory activities against viral influenza A (H1N1) and bacterial Vibrio cholerae neuraminidase (VCNA). Compared to the positive controls, all tested polyphenols displayed a weak inhibition of the viral enzyme but similar or even higher potency on the bacterial neuraminidase. Structure–activity relationship analyses revealed the presence of galloyl groups and the hydroxylation pattern of the flavan skeleton to be crucial for inhibitory activity. The combination of zanamivir and EPs® 7630 (root extract of Pelargonium sidoides) showed synergistic inhibitory effects on the bacterial neuraminidase. Co-crystal structures of VCNA with oseltamivir carboxylate and zanamivir provided insight into bacterial versus viral enzyme-inhibitor interactions. The current data clearly indicate that inhibitor potency strongly depends on the biological origin of the enzyme and that results are not readily transferable. The therapeutic relevance of our findings is briefly discussed.
... For example, garlic has sulfur-containing compounds such as alliin, allicin, diallyl sulfide, diallyl disulfide, diallyl trisulfide, ajoene, and S-allylcysteine [31] which may react with the thiol groups of disulfide bonds and cause conformational changes of the HA receptors, thus making the virus unable to interact with the host cell surface. In addition, honey [32] and ginger [33] contains hydrogen peroxide, which can act as a potent antimicrobial agent. ...
Three influenza A virus isolates, one from a human, one from a chicken, and one from a wild bird dropping, were used to infect human peripheral blood mononuclear cells (PBMCs) in culture. The influenza strains were H1N, H5/H7-N1/N2 (mixture) and H9N, respectively and were assessed in vitro in human PBMCs in the presence of Phytohemagglutinin (PHA). Viral replication was estimated by visual cytopathic effect (CPE). H1N2 CPE included budding of lymphocytes, fusion of infected cells with neighboring lymphocytes, and syncytial formation. H5/H7- N1/N2 and H9N2 each caused CPE that included bulging of cells with large vacuoles. The supernatant of infected lymphocyte culture was used to infect MDCK cell lines. Influenza viral RNA was detected in extracts of MDCK cell lines and from lymphocytes infected with each of the three virus strains as judged by RT-PCR, confirming that all three isolates were able to replicate in human lymphocytes in vitro. The antiviral activity of a mixture of honey, ginger, and garlic (HGG) extracts, an over the counter drug commonly used in Pakistan to treat patients with influenza virus, was compared to the antiviral drug amantadine for its ability to decrease replication of the H1N2 strain in human PBMCs in vitro. HGG significantly inhibited H1N2 virus growth as judged by CPE, haemagglutination assays, and qRT-PCR. Interestingly, HGG also appeared to promote proliferation of human lymphocytes. These finding suggest that a mixture of crude extracts from honey, ginger, and garlic may be clinically useful against influenza virus.
The benefits of honey have been recognized since ancient times for treating numerous diseases. However, in today's modern era, the use of traditional remedies has been rapidly diminishing due to the complexities of modern lifestyles. While antibiotics are commonly used and effective in treating pathogenic infections, their inappropriate use can lead to the development of resistance among microorganisms, resulting in their widespread prevalence. Therefore, new approaches are constantly required to combat drug-resistant microorganisms, and one practical and useful approach is the use of drug combination treatments.
Manuka honey, derived from the manuka tree (Leptospermum scoparium) found exclusively in New Zealand, has garnered significant attention for its biological potential, particularly due to its antioxidant and antimicrobial properties. Moreover, when combined with antibiotics, it has demonstrated the ability to enhance their effectiveness. In this review, we delve into the chemical markers of manuka honey that are currently known, as well as detail the impact of manuka honey on the management of infectious diseases up to the present.
We report the structural functionalization of the terminal amino group of N¹‐(7‐chloroquinolin‐4‐yl) butane‐1,4‐diamine, leading to a series of 7‐chloro‐4‐aminoquinoline derivatives, and their evaluation as potent anti‐malarial and anti‐viral agents. Some compounds exhibited promising anti‐malarial effects against the Plasmodium falciparum 3D7 (chloroquine‐sensitive) and Dd2 (chloroquine‐resistant) strains. In addition, these compounds were assayed in vitro against influenza A virus (IAV) and severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2). Compound 5 h, bearing an N‐mesityl thiourea group, displayed pronounced anti‐infectious effects against malaria, IAV, and SARS‐CoV‐2. These results provide new insights into drug discovery for the prevention or treatment of malaria and virus co‐infection.
The COVID-19 pandemic is a major threat worldwide. Since it is a contagious disease and there are no established treatment procedures, the situation demands stronger preventive measures. Thus, an effective and reliable prevention method is important. The use of gargles and nasal rinses are well-known ancient Ayurvedic procedures that have been an effective weapon against many infections related to the throat and nasal path. Thus, their role in preventive management of COVID-19 should be investigated. Soaps and detergents are proven to be very effective in destroying the virus outside the body, but can this be useful in the throat? Most of the virus passes to the lungs via the throat and nasal route, so inactivation or flushing out of the virus from the throat and nasal path itself may prevent its entry to the lungs. The use of gargles and nasal rinses consisting of saponins as natural surfactants may provide antiviral action. The virus is said to remain in the throat for 3 to 4 days. Gargling allows these natural surfactants to come directly into contact with the adhering virus in the throat, thereby flushing out or inactivating the virus. Many studies have shown that constituents like glycyrrhizin (liquorice), curcumin (turmeric) and methylglyoxal (manuka honey) etc. have potential activity against life threatening viruses. Thus, a medicated gargle and nasal rinse incorporating these natural surfactants along with potential antiviral drugs may be able to exert an acceptable result in preventive management of SARS-CoV-2.
To explore potential biological activities of trifluoromethyl heterocycles, we recently developed a synthetic approach to assess a series of α‐trifluoromethyl‐α,β‐unsaturated lactones and trifluoromethyl pyrazolinones. The compounds were tested for their antimicrobial activity, and we found that some compounds had anti‐influenza viral activity. The β‐aryl‐α‐trifluoromethyl α,β‐unsaturated lactone derivatives 5g, 7b and the trifluoromethyl pyrazolinone 12c possessed promising inhibitory activity against influenza virus type A, strain A/WSN/33 (H1N1). These hit compounds 5g, 7b, and 12c were successfully optimized, and we identified that the most potent compound 5h showed inhibitory activity against various types of influenza A and B viruses in the low‐micromolar range without showing cytotoxicity. Moreover, 5h was more effective against the clinical isolate A/California/7/2009 (H1N1pdm) strain than the influenza viral polymerase inhibitor, favipiravir (T‐705). We also delineated the structure–activity relationship and obtained mechanistic insights into inhibition of the viral polymerase.
Micellization of di-n-decyldimethylammonium chloride, [DiC10][Cl], and octaethylene glycol monododecyl ether, C12E8, mixtures have been investigated by surface tension and conductivity measurements. From these results, various physicochemical and thermodynamic key parameters (e.g. micellar mole fraction of [DiC10][Cl], interaction parameter, free energy of micellization, etc.) have been evaluated and discussed in detail. The results prove high synergistic effect between the two surfactants. Based on these results, the virucidal activity of an equimolar mixture of [DiC10][Cl] and C12E8 has been investigated. A marked synergism was observed on lipid-containing deoxyribonucleic and ribonucleic acid viruses, such as herpes virus, respiratory syncytial virus, and vaccinia viruses. In contrast, Coxsackievirus (non-enveloped virus) was not inactivated. These results support that the mechanism is based on the extraction of lipids and/or proteins from the envelope inside the mixed micelles. This extraction creates "holes" the size of which increases with concentration up to a specific value which triggers the virus inactivation. Such a mixture could be used to extend the spectrum of virucidal activity of the amphiphiles virucides commonly employed in numerous disinfectant solutions.
Neuraminidase inhibitors (NAIs) play a major role for managing influenza infections. The widespread oseltamivir resistance among 2007-08 seasonal A(H1N1) viruses and community outbreaks of oseltamivir-resistant A(H1N1)pdm09 strains highlight the need for additional anti-influenza agents. Laninamivir is a novel long-lasting NAI that has demonstrated in vitro activity against influenza A and B viruses and its prodrug (laninamivir octanoate) is in phase II clinical trials in US and other countries. Currently, little information is available on the mechanisms of resistance to laninamivir. In this study, we first performed neuraminidase (NA) inhibition assays to determine the activity of laninamivir against a set of influenza A viruses containing NA mutations conferring resistance to one or many other NAIs. We also generated drug-resistant A(H1N1) and A(H3N2) viruses under in vitro laninamivir pressure. Laninamivir demonstrated a profile of susceptibility that was similar to that of zanamivir. More specifically, it retained activity against oseltamivir-resistant H275Y and N295S A(H1N1) mutantsand the E119V A(H3N2) variant. In vitro, laninamivir pressure selected the E119A NA substitution in the A/Solomon Islands/3/2006 A(H1N1) background whereas E119K and G147E NA changes along with a K133E HA substitution were selected in the A/Quebec/144147/2009 A(H1N1)pdm09 strain. In the A/Brisbane/10/2007 A(H3N2) background, a large NA deletion accompanied with S138A/P194L HA substitutions were selected. This H3N2 variant had altered receptor-binding properties and was highly resistant to laninamivir in plaque reduction assays. Overall, we confirmed the similarity between zanamivir and laninamivir susceptibility profiles and demonstrated that both NA and HA changes can contribute to laninamivir resistance in vitro.
Influenza viruses collected from regions of Asia, Africa and Oceania between 2009 and 2012 were tested for their susceptibility to two new neuraminidase inhibitors, peramivir and laninamivir. All viruses tested had normal laninamivir inhibition. However, 3·2% (19/599) of A(H1N1)pdm09 viruses had highly reduced peramivir inhibition (due to H275Y NA mutation) and <1% (6/1238) of influenza B viruses had reduced or highly reduced peramivir inhibition, with single occurrence of variants containing I221T, A245T, K360E, A395E, D432G and a combined G145R+Y142H mutation. These data demonstrate that despite an increase in H275Y variants in 2011, there was no marked change in the frequency of peramivir- or laninamivir-resistant variants following the market release of the drugs in Japan in 2010.
Influenza virus infection is a major public health problem that leads to significant morbidity and mortality. The emergence of resistance to the currently available anti-influenza agents has necessitated the development of new drugs with novel targets. Studying known ethno-medicinal plants is a promising approach for the discovery of new antiviral compounds. Alchemilla mollis is used in traditional medicine in Europe for different indications, including minimizing the symptoms of a sore throat. In this study, we found that A. mollis extract has anti-influenza activity, and investigated the mechanism underlying its inhibition of influenza virus replication. Plaque assays demonstrated that treatment of cells with A. mollis extract prior to infection did not inhibit influenza virus infection. However, plaque formation was markedly reduced when infected cells were overlaid with an agarose gel containing A. mollis extract. In addition, exposure of the virus to A. mollis extract prior to infection and treatment of cells during virus infection significantly suppressed plaque formation. Influenza virus-induced hemagglutination of chicken red blood cells was inhibited by A. mollis extract treatment. The inhibitory effect was observed against influenza A virus subtypes H1N1, H3N2, and H5N2. These findings suggest that A. mollis extract has virucidal or neutralizing activity against influenza virus particles. Furthermore, inhibitory effect of zanamivir synergistically increased after combination with A. mollis extract. Our results suggest that A. mollis extract has the potential to be developed as an antiinfluenza agent.
Avian influenza A viruses rarely infect humans; however, when human infection and subsequent human-to-human transmission occurs, worldwide outbreaks (pandemics) can result. The recent sporadic infections of humans in China with a previously unrecognized avian influenza A virus of the H7N9 subtype (A(H7N9)) have caused concern owing to the appreciable case fatality rate associated with these infections (more than 25%), potential instances of human-to-human transmission, and the lack of pre-existing immunity among humans to viruses of this subtype. Here we characterize two early human A(H7N9) isolates, A/Anhui/1/2013 (H7N9) and A/Shanghai/1/2013 (H7N9); hereafter referred to as Anhui/1 and Shanghai/1, respectively. In mice, Anhui/1 and Shanghai/1 were more pathogenic than a control avian H7N9 virus (A/duck/Gunma/466/2011 (H7N9); Dk/GM466) and a representative pandemic 2009 H1N1 virus (A/California/4/2009 (H1N1pdm09); CA04). Anhui/1, Shanghai/1 and Dk/GM466 replicated well in the nasal turbinates of ferrets. In nonhuman primates, Anhui/1 and Dk/GM466 replicated efficiently in the upper and lower respiratory tracts, whereas the replicative ability of conventional human influenza viruses is typically restricted to the upper respiratory tract of infected primates. By contrast, Anhui/1 did not replicate well in miniature pigs after intranasal inoculation. Critically, Anhui/1 transmitted through respiratory droplets in one of three pairs of ferrets. Glycan arrays showed that Anhui/1, Shanghai/1 and A/Hangzhou/1/2013 (H7N9) (a third human A(H7N9) virus tested in this assay) bind to human virus-type receptors, a property that may be critical for virus transmissibility in ferrets. Anhui/1 was found to be less sensitive in mice to neuraminidase inhibitors than a pandemic H1N1 2009 virus, although both viruses were equally susceptible to an experimental antiviral polymerase inhibitor. The robust replicative ability in mice, ferrets and nonhuman primates and the limited transmissibility in ferrets of Anhui/1 suggest that A(H7N9) viruses have pandemic potential.
Recently renewed interest in the therapeutic properties of honey has led to the search for new antimicrobial honeys. This study was undertaken to assess the antimicrobial activity and composition of a locally produced Portobello honey (PBH) on three bacteria known to infect wounds. Manuka honey (MH) was used for comparative purposes. Broth culture and agar disc diffusion assays were used to investigate the antimicrobial properties of honey. The honeys were tested at four concentrations: 75%, 50%, 10% and 1% (v/v) and compared with an untreated control. The composition of honey was determined by measuring: polyphenol content by Folin Ciocalteau method, antioxidant capacity by ferric ion reducing power assay, hydrogen peroxide (H(2) O(2) ) by catalase test, pH and sugar content by pH strips and refractometer, respectively. Both honeys at 75% and 50% inhibited the majority of the three bacteria tested. 10% PBH exhibited antimicrobial activity to the lesser extent than 10% MH. The difference was very significant (p ≤ 0.001). Both honeys were acidic with pH 4, and both produced H(2) O(2) . The sugar content of PBH was higher than MH, but the difference was not significant. The MH had significantly higher levels of the polyphenols and antioxidant activity than PBH. Copyright © 2012 John Wiley & Sons, Ltd.
Pseudomonas aeruginosa (PA) and Staphylococcus aureus (SA) biofilms are associated with poor chronic rhinosinusitis (CRS) disease control following surgery. Manuka honey (MH) has been shown to be both an effective in vitro treatment agent for SA and PA biofilms and nontoxic to sinonasal respiratory mucosa. Methylglyoxal (MGO) has been reported to be the major antibacterial agent in MH. The effect of this agent against SA and PA biofilms has yet to be reported. Our objective was to determine the in vitro effect of MGO against biofilms of SA and PA, via in vitro testing of MGO against bacterial biofilms.
An established biofilm model was used to determine the effective concentration (EC) of MGO against 10 isolates of methicillin-resistant SA (MRSA) and PA. The EC of MGO was also determined against planktonic (free-swimming) MRSA and PA.
For MRSA, the EC against planktonic organisms was a concentration of 0.08 mg/mL to 0.3 mg/mL whereas against the biofilm MRSA isolates, the EC ranged from 0.5 mg/mL to 3.6 mg/mL. For PA, the EC against planktonic organisms was a concentration of 0.15 mg/mL to 1.2 mg/mL for planktonic organisms whereas against the biofilm PA isolates, the EC ranged from 1.8 mg/mL to 7.3 mg/mL.
MGO, a component of MH, is an effective antimicrobial agent against both planktonic and biofilm MRSA and PA organisms in vitro.
Two neuraminidase (NA) inhibitors, zanamivir (Relenza) and oseltamivir phosphate (Tamiflu), have been licensed for use for
the treatment and prophylaxis of influenza. We have reported on laninamivir (code name, R-125489), a novel neuraminidase inhibitor,
and have discovered that the laninamivir prodrug CS-8958 worked as a long-acting neuraminidase inhibitor in a mouse influenza
virus infection model when it is intranasally administered. In this study, CS-8958 was administered just once 7 days before
infection and showed significant efficacy in vivo. The efficacy of a single administration of CS-8958 after viral infection was then compared with that of repeated administrations
of oseltamivir phosphate or zanamivir in mice and ferrets. CS-8958 showed efficacy superior or similar to the efficacies of
the two licensed NA inhibitors. CS-8958 also significantly reduced the titers of an oseltamivir-resistant H1N1 virus with
a neuraminidase H274Y substitution in a mouse infection model. These results suggest that since CS-8958 is characteristically
long lasting in the lungs, it may be ideal for the prophylaxis and treatment of influenza.
Antiviral drug resistance for influenza therapies remains a concern due to the high prevalence of H1N1 2009 seasonal influenza isolates which display H274Y associated oseltamivir-resistance. Furthermore, the emergence of novel H1N1 raises the potential that additional reassortments can occur, resulting in drug resistant virus. Thus, additional antiviral approaches are urgently needed. DAS181 (Fludase), a sialidase fusion protein, has been shown to have inhibitory activity against a large number of seasonal influenza strains and a highly pathogenic avian influenza (HPAI) strain (H5N1). Here, we examine the in vitro activity of DAS181 against a panel of 2009 oseltamivir-resistant seasonal H1N1 clinical isolates. The activity of DAS181 against nine 2009, two 2007, and two 2004 clinical isolates of seasonal IFV H1N1 was examined using plaque number reduction assay on MDCK cells. DAS181 strongly inhibited all tested isolates. EC50 values remained constant against isolates from 2004, 2007, and 2009, suggesting that there was no change in DAS181 sensitivity over time. As expected, all 2007 and 2009 isolates were resistant to oseltamivir, consistent with the identification of the H274Y mutation in the NA gene of all these isolates. Interestingly, several of the 2007 and 2009 isolates also exhibited reduced sensitivity to zanamivir, and accompanying HA mutations near the sialic acid binding site were observed. DAS181 inhibits IFV that is resistant to NAIs. Thus, DAS181 may offer an alternative therapeutic option for seasonal or pandemic IFVs that become resistant to currently available antiviral drugs.
The recurring emergence of influenza virus strains that are resistant to available antiviral medications has become a global health concern, especially in light of the potential for a new influenza virus pandemic. Currently, virtually all circulating strains of influenza A virus in the United States are resistant to either of the two major classes of anti-influenza drugs (adamantanes and neuraminidase inhibitors). Thus, new therapeutic approaches that can be rapidly deployed and that will address the issue of recurring resistance should be developed. We have tested double and triple combinations of the approved anti-influenza drugs oseltamivir and amantadine together with ribavirin against three influenza virus strains using cytopathic effect inhibition assays in MDCK cells. We selected A/New Caledonia/20/99 (H1N1) and A/Sydney/05/97 (H3N2) as representatives of the wild-type versions of the predominant circulating seasonal influenza virus strains and A/Duck/MN/1525/81 (H5N1) as a representative of avian influenza virus strains. Dose-response curves were generated for all drug combinations, and the degree of drug interaction was quantified using a model that calculates the synergy (or antagonism) between the drugs in double and triple combinations. This report demonstrates that a triple combination of antivirals was highly synergistic against influenza A virus. Importantly, the synergy of the triple combination was 2- to 13-fold greater than the synergy of any double combination depending on the influenza virus subtype. These data support the investigation of a novel combination of oseltamivir, amantadine, and ribavirin as an effective treatment for both seasonal and pandemic influenza virus, allowing the efficient use of the existing drug supplies.
A Kegin-type polyoxometalate, PM-523, in combination with ribavirin, was tested for its therapeutic effectiveness against influenza virus (FluV) A (H1N1) infection in tissue culture and in mice. PM-523 [(PriNH3)6H [PTi2W10O38(O2)2] x H2O, where Pri is isopropanol] and ribavirin individually inhibited FluV A-induced cytopathic effects in Madin-Darby canine kidney (MDCK) cells at median effective concentrations (EC50s) of 30 and 34 microM, respectively, and at 70% effective concentrations (EC70s) of 48 and 72 microM, respectively. On the other hand, a combination of PM-523 and ribavirin at a ratio of 1:16 exhibited lower EC50s and EC70s than each compound used singly, and combination indices were less than 1. A wide range of combinations of PM-523 and ribavirin at ratios of from 1:128 to 1:1 exhibited additive or synergistic anti-FluV effects in MDCK cells. When these compounds were tested for their anti-FluV A activities in vivo by aerosol exposure of mice which had been infected with a lethal dose of FluV A by an intranasal route, a 1:16 combination of PM-523 and ribavirin was found to have a significantly better therapeutic effect than a single dose of either compound used singly with respect to both the survival rate of the mice and the virus titer in the lungs of the infected mice. PM-523 was effective for the treatment of experimental FluV infection, and in combination with ribavirin, PM-523 exhibited enhanced anti-FluV effects in vitro and in vivo compared with the effect of PM-523 alone.
Neuraminidase promotes influenza virus release from infected cells and facilitates virus spread within the respiratory tract. Several potent and specific inhibitors of this enzyme have been developed, and two (zanamivir and oseltamivir) have been approved for human use. Unlike amantadine and rimantadine that target the M2 protein of influenza A viruses, these drugs inhibit replication of both influenza A and B viruses. Zanamivir is delivered by inhalation because of its low oral bioavailability whereas oseltamivir is administered by mouth. Early treatment with either drug reduces the severity and duration of influenza symptoms and associated complications. Both agents are effective for chemoprophylaxis. Because of a broader antiviral spectrum, better tolerance, and less potential for emergence of resistance than is seen with the M2 inhibitors, the neuraminidase inhibitors represent an important advance in the treatment of influenza.
There is insufficient information about combination therapy with approved anti-influenza agents. We tested combinations that paired a neuraminidase (NA) inhibitor (zanamivir, oseltamivir carboxylate, or peramivir) with rimantadine against infection of MDCK cells with H1N1 and H3N2 subtypes of influenza A virus and characterized their mode of interaction. When reduction of extracellular virus was analyzed by individual regression models and three-dimensional representations of the data, all three combinations showed additive and synergistic effects with no cytotoxicity. Maximum synergy against A/New Caledonia/20/99 (H1N1) virus infection was observed with <2.5 microM rimantadine paired with low concentrations of NA inhibitors. All combinations reduced the extracellular yield of A/Panama/2007/99 (H3N2) influenza virus synergistically. However, our findings were different for the cell-associated virus yield. At some drug concentrations, the yield of cell-associated virus was inhibited antagonistically. Therefore, the method of analysis can be a crucial factor in evaluating the interactions of drugs with different mechanisms. We hypothesize that assays based on cell-associated virus yield may underestimate the efficacies of drug combinations that include an NA inhibitor. Taken together, our results suggest that regimens that combine NA inhibitors and rimantadine exert synergistic anti-influenza effects in vitro. These findings provide baseline information for therapeutic testing of the drug combinations in vivo.
Influenza A and B viruses are enveloped negative strand RNA viruses that contain eight RNA segments. Segment 7 encodes the M1 protein and a proton channel protein (A/M2 for influenza A virus, BM2 for influenza B virus). A/M2 and BM2 proteins have very different amino sequences, except they share a common motif, HXXXW, in their transmembrane domain, which is essential for channel activation and gating. Channel activities of A/M2 and BM2 proteins are essential to virus replication. A/M2 protein is also the target of the adamantane class of drugs (amantadine and its analog compound rimantadine); one of two antiviral drugs against influenza A virus, whereas the BM2 protein is not inhibited by amantadine. Since the identification of the A/M2 protein in 1981, it has been extensively studied by mutagenesis, electrophysiology, and numerous structural studies, such as CD, fluorescence, solid state nuclear magnetic resonance (NMR), solution NMR, and X-ray crystallography. In this chapter, we focus on A/M2 protein to discuss recent advances in structure-function studies and the mechanism for drug inhibition. Finally, ongoing studies to develop an antiviral drug against the currently circulating form of the A/M2 protein are summarized.
The influenza virus M2 protein was expressed in Xenopus laevis oocytes and shown to have an associated ion channel activity selective for monovalent ions. The anti-influenza virus drug amantadine hydrochloride significantly attenuated the inward current induced by hyperpolarization of oocyte membranes. Mutations in the M2 membrane-spanning domain that confer viral resistance to amantadine produced currents that were resistant to the drug. Analysis of the currents of these altered M2 proteins suggests that the channel pore is formed by the transmembrane domain of the M2 protein. The wild-type M2 channel was found to be regulated by pH. The wild-type M2 ion channel activity is proposed to have a pivotal role in the biology of influenza virus infection.
Background and aims:
Influenza viruses are a serious threat to human health and cause thousands of deaths annually. Thus, there is an urgent requirement for the development of novel anti-influenza virus drugs. Therefore, the aim of this study was to evaluate the anti-influenza viral activity of honey from various sources.
Methods:
Antiviral activities of honey samples were evaluated using MDCK cells. To elucidate the possible mechanism of action of honey, plaque inhibition assays were used. Synergistic effects of honey with known anti-influenza virus drugs such as zanamivir or oseltamivir were tested.
Results:
Manuka honey efficiently inhibited influenza virus replication (IC50 = 3.6 ± 1.2 mg/mL; CC50 = 82.3 ± 2.2 mg/mL; selective index = 22.9), which is related to its virucidal effects. In the presence of 3.13 mg/mL manuka honey, the IC50 of zanamivir or oseltamivir was reduced to nearly 1/1000th of their single use.
Conclusions:
Our results showed that honey, in general, and particularly manuka honey, has potent inhibitory activity against the influenza virus, demonstrating a potential medicinal value.
In addition to immunization programs, antiviral agents can play a major role for the control of seasonal influenza epidemics and may also provide prophylactic and therapeutic benefits during an eventual pandemic. The purpose of this article is to review the mechanism of action, pharmacokinetics and clinical indications of neuraminidase inhibitors (NAIs) with an emphasis on the emergence of antiviral drug resistance. There are two approved NAIs compounds in US: inhaled zanamivir and oral oseltamivir, which have been commercially available since 1999-2000. In addition, two other NAIs, peramivir (an intravenous cyclopentane derivative) and laninamivir (a long-acting NAI administered by a single nasal inhalation) have been approved in certain countries and are under clinical evaluations in others. As for other antivirals, the development and dissemination of drug resistance is a significant threat to the clinical utility of NAIs. The emergence and worldwide spread of oseltamivir-resistant seasonal A(H1N1) viruses during the 2007-09 seasons emphasize the need for continuous monitoring of antiviral drug susceptibilities. Further research priorities should include a better understanding of the mechanisms of resistance to existing antivirals, the development of novel compounds which target viral or host proteins and the evaluation of combination therapies for improved treatment of severe influenza infections, particularly in immunocompromised individuals. This article forms part of a symposium in Antiviral Research on "Treatment of influenza: targeting the virus or the host".
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.
Preliminary screening data indicate that six types of aliphatic compounds are highly active against Newcastle disease and influenza viruses in the embryonated egg. These compounds include certain: (1) α-ketoaldehydes, (2) α-hydroxyaldehydes, (3) vicinal triketones, (4) cyclic 1,2-diketones, (5) α-keto primary alcohols and (6) enediols. The preparation of some of these compounds and their derivatives is described.
The half maximal inhibitory concentration (IC(50)) of four neuraminidase inhibitors (NAIs), oseltamivir, zanamivir, laninamivir, and peramivir; was measured using influenza viruses isolated in the 2010-2011 influenza season in Japan. Clinical samples for viral isolation were obtained from nasal aspiration, nasopharyngeal swab, or self-blown nasal discharge and cultured with Madin-Darby canine kidney cells. The type and subtype of H3N2 or B were determined by reverse transcriptase polymerase chain reaction (RT-PCR). For the A(H1N1)pdm09 virus, the subtype was determined by real-time RT-PCR. IC(50)s to oseltamivir carboxylate, zanamivir, laninamivir, and peramivir were determined by a fluorescence-based neuraminidase inhibition assay. Influenza viruses were isolated from 269 patients. A(H1N1)pdm09, H3N2, and B were isolated from 185, 54, and 30 patients, respectively. The geometric means of IC(50) for oseltamivir were 0.86 and 0.73 nM to A (H1N1) pdm09, except for the two outlier viruses described below and H3N2, respectively, and 33.12 nM for B. The geometric means of IC(50) for the other three NAIs were lowest to A(H1N1)pdm09 and highest to B. Two A(H1N1)pdm09 isolates showed very high IC(50) values for oseltamivir (840 and 600 nM) and peramivir (19 and 24 nM). No isolate showed significantly high IC(50) values for zanamivir or laninamivir. Continuous surveillance against the emergence or spread of influenza virus with high IC(50) values for anti-influenza drugs is important.
Isolation and determination of the nucleotide sequence of hemagglutinin (HA) of the pandemic (H1N1) 2009 influenza viruses found in Nagasaki, Japan, were conducted. The alignment results of the predicted HA amino acid sequences of these strains compared to the known global isolates revealed 5 specific amino acid differences located within the antigenic sites. The phylogenetic analyses revealed that the majority of the Nagasaki isolates could be classified into 6 phylogenetic clusters. Almost all isolates collected in the early season were classified into cluster I, which apparently originated from A/Nagasaki/HA-6/2009 isolated from a patient who returned from the Philippines. This cluster ceased to spread after November 2009. Between the end of August 2009 and January 2010, 5 new phylogenetic clusters (II-VI) emerged with viruses from different origins, and cluster III continuously advanced until March 2010. These results suggest that the onset of the influenza epidemic in Nagasaki originated from patient(s) who returned from the Philippines, and subsequently, various imported strains from different origins sustained the virus spread. Among the Nagasaki isolates, A/Nagasaki/HA-58/2009 having an H275Y mutation in the neuraminidase gene, which confers resistance to oseltamivir, was isolated. This is the first report in which an oseltamivir-resistant pandemic H275Y mutant was identified in Nagasaki Prefecture.
Influenza epidemics cause numerous deaths and millions of hospitalizations each year. Because of the alarming emergence of resistance to anti-influenza drugs, there is a need to identify new naturally occurring antiviral molecules. We tested the hypothesis that pomegranate polyphenol extract (PPE) has anti-influenza properties. Using real time PCR, plaque assay, and TCID 50% hemagglutination assay, we have shown that PPE suppresses replication of influenza A virus in MDCK cells. PPE inhibits agglutination of chicken red blood cells (cRBC) by influenza virus and is virucidal. The single-cycle growth conditions indicated that independent of the virucidal effect PPE also inhibits viral RNA replication. PPE did not alter virus ribonucleoprotein (RNP) entry into nucleus or translocation of virus RNP from nucleus to cytoplasm in MDCK cells. We evaluated four major Polyphenols in PPE (ellagic acid, caffeic acid, luteolin, and punicalagin) and demonstrated that punicalagin is the effective, anti-influenza component of PPE. Punicalagin blocked replication of the virus RNA, inhibited agglutination of chicken RBC's by the virus and had virucidal effects. Furthermore, the combination of PPE and oseltamivir synergistically increased the anti-influenza effect of oseltamivir. In conclusion, PPE inhibited the replication of human influenza A/Hong Kong (H3N2) in vitro. Pomegranate extracts should be further studied for therapeutic and prophylactic potential especially for influenza epidemics and pandemics.
Oseltamivir, a specific influenza neuraminidase inhibitor, is an effective treatment for seasonal influenza. Emergence of drug-resistant influenza viruses after treatment has been reported, particularly in children in Japan, where the dosing schedule is different from that used throughout the rest of the world. We investigated the emergence of drug-resistant infection in children treated with a tiered weight-based dosing regimen.
We analyzed sequential clinical nasopharyngeal samples, obtained before and after tiered weight-based oseltamivir therapy, from children with acute influenza during 2005-2007. We isolated viruses, tested for drug resistance with use of a fluorescence-based neuraminidase inhibition assay, performed neuraminidase gene sequencing, and determined quantitative viral loads.
Sixty-four children (34 with influenza A subtype H3N2, 11 with influenza A subtype H1N1, and 19 with influenza B virus) aged 1-12 years (median age, 3 years, 1 month) were enrolled. By days 4-7 after initiation of treatment, of 64 samples tested, 47 (73.4%) and 26 (40.6%) had virus detectable by reverse-transcriptase polymerase chain reaction and culture, respectively. By days 8-12 after initiation of treatment, of 53 samples tested, 18 (33.9%) and 1 (1.8%) had virus detectable by reverse-transcriptase polymerase chain reaction and culture, respectively. We found no statistically significant differences in the reduction of viral shedding or time to clearance of virus between viral subtypes. Antiviral-resistant viruses were recovered from 3 (27.3%) of 11 children with influenza A subtype H1N1, 1 (2.9%) of 34 children with influenza A subtype H3N2, and 0 (0%) of 19 children with influenza B virus, all of whom were treated with oseltamivir (P = .004). There was no evidence of prolonged illness in children infected with drug-resistant virus.
Drug resistance emerges at a higher rate in influenza A subtype H1N1 virus than in influenza A subtype H3N2 or influenza B virus after tiered weight-based oseltamivir therapy. Virological surveillance for patterns of drug resistance is essential for determination of antiviral treatment strategies and for composition of pandemic preparedness stockpiles.
The inactivating effect of methyl glyoxal on foot-and-mouth disease (FMD) virus was studied. The rate of inactivation depended upon the drug concentration, incubation temperature and pH of the medium. RNA recovered from the inactivated virus was not infectious. The complement-fixing activity of the virus was not reduced by methyl glyoxal treatment. The antigenicity of inactivated virus preparations determined by levels of virus neutralizing antibody in the blood sera of immunized white rats and rabbits was not inferior to that of the initial uninactivated virus.
The influenza virus M2 protein was expressed in Xenopus laevis oocytes and shown to have an associated ion channel activity selective for monovalent ions. The anti-influenza virus drug amantadine hydrochloride significantly attenuated the inward current induced by hyperpolarization of oocyte membranes. Mutations in the M2 membrane-spanning domain that confer viral resistance to amantadine produced currents that were resistant to the drug. Analysis of the currents of these altered M2 proteins suggests that the channel pore is formed by the transmembrane domain of the M2 protein. The wild-type M2 channel was found to be regulated by pH. The wild-type M2 ion channel activity is proposed to have a pivotal role in the biology of influenza virus infection.
Serial dilutions of Clostridium difficile culture filtrates were incubated overnight with HeLa cell monolayers. Cells were fixed in formalin, stained with crystal violet, rinsed, and drained. Cell rounding could be observed microscopically in the stained monolayers. Absorbance of the retained dye on monolayers in the drained wells was measured at 595 nm-405 nm. End points could also be estimated visually. The dilution at which dye absorbance was reduced by 50% agreed with that determined by microscopic observations. Five replicate dilution series showed high reproducibility. Specificity was verified by neutralisation with crude rabbit antibody to C difficile toxins. Cytotoxicity in faecal specimens was assayed in the same way, allowing reporting of titres, comparison with standard toxin preparations, and determination of the extent of neutralisation to be made. This novel assay technique has proved effective and reliable in a clinical setting and should allow the gathering of more information on the epidemiology of antibiotic associated colitis.
The determination of the 3-dimensional structure of the influenza virus neuraminidase in 1983 has served as a platform for understanding interactions between antibodies and protein antigens, for investigating antigenic variation in influenza viruses, and for devising new inhibitors of the enzyme. That work is reviewed here, together with more recent developments that have resulted in one of the inhibitors entering clinical trials as an anti-influenza virus drug.
In May, 1997, a 3-year-old boy in Hong Kong was admitted to the hospital and subsequently died from influenza pneumonia, acute respiratory distress syndrome, Reye's syndrome, multiorgan failure, and disseminated intravascular coagulation. An influenza A H5N1 virus was isolated from a tracheal aspirate of the boy. Preceding this incident, avian influenza outbreaks of high mortality were reported from three chicken farms in Hong Kong, and the virus involved was also found to be of the H5 subtype.
We carried out an antigenic and molecular comparison of the influenza A H5N1 virus isolated from the boy with one of the viruses isolated from outbreaks of avian influenza by haemagglutination-inhibition and neuraminidase-inhibition assays and nucleotide sequence analysis.
Differences were observed in the antigenic reactivities of the viruses by the haemagglutination-inhibition assay. However, nucleotide sequence analysis of all gene segments revealed that the human virus A/Hong Kong/156/97 was genetically closely related to the avian A/chicken/Hong Kong/258/97.
Although direct contact between the sick child and affected chickens has not been established, our results suggest transmission of the virus from infected chickens to the child without another intermediate mammalian host acting as a "mixing vessel". This event illustrates the importance of intensive global influenza surveillance.
Four different methods for interpreting the results of checkerboard synergy testing were compared by applying each to a set of synergy study data. Statistically significant differences in synergy were detected among methods (% synergy ranged from 10 to 83%). As interpretations were found to vary widely based upon method, one should be aware of this in interpreting the relevant literature.
At present, three licensed antiviral influenza agents are available in Japan: amantadine, zanamivir, and oseltamivir. These antiviral agents can be used for controlling and preventing influenza, but they are not a substitute for vaccination. Amantadine is an antiviral drug with activity against influenza A viruses, but not influenza B viruses. Persons who have influenza A infection and who are treated with amantadine can shed sensitive viruses early in the course of treatment and later shed drug-resistant viruses, especially after 5-7 days of therapy. Such persons can benefit from therapy even when resistant viruses emerge. In screening for amantadine susceptibility, enzyme-linked immunoassays, plaque reduction assays, and TCID50/0.2 ml titration are employed. The molecular changes associated with resistance have been identified as single-nucleotide changes, leading to corresponding amino acid substitutions in one of four critical sites, amino acids 26, 27, 30, and 31, in the transmembrane region of the M2 protein. The polymerase chain reaction (PCR)-restriction fragment length polymorphism analysis method is quite useful. Resistant viruses have been circulated in outbreak situations at nursing homes where amantadine was used not only for treating influenza virus infection but also for Parkinson's disease. Measures should be taken to reduce contact, as much as possible, between persons taking and those not taking antiviral drugs for treatment or chemoprophylaxis.
Atazanavir, an azapeptide protease inhibitor (PI), has pharmacokinetics that allow once-daily dosing, and it is not associated with significant PI-associated dyslipidemia.
A randomized, double-blind, double-dummy, active-controlled, 2-arm study comparing the antiviral efficacy and safety of atazanavir 400 mg administered once daily with efavirenz 600 mg administered once daily in combination with open-label fixed-dose zidovudine plus lamivudine twice daily. The 810 treatment-naive patients were stratified by HIV RNA level. The primary efficacy end point was the proportion of treated patients with HIV RNA levels <400 copies/mL through week 48.
At week 48, HIV RNA levels were <400 copies/mL in 70% of patients receiving atazanavir and 64% of patients receiving efavirenz (intent-to-treat, difference; 95% confidence interval: 5.2%; -1.2%, 11.7%). Median CD4 cell counts increased at comparable magnitudes and rates in the 2 treatment arms (mean change at week 48: 176 cells/mm with atazanavir, 160 cells/mm with efavirenz). Atazanavir-treated patients relative to comparator-treated patients did not demonstrate significant increases in total cholesterol, fasting low-density lipoprotein cholesterol, or fasting triglycerides over 48 weeks of therapy. Atazanavir-linked bilirubin elevations infrequently resulted in treatment discontinuation (<1%). Atazanavir treatment did not increase fasting glucose or insulin levels.
For initial HIV treatment, a highly active antiretroviral therapy regimen of atazanavir/zidovudine/lamivudine is as efficacious and well tolerated as the combination of efavirenz/zidovudine/lamivudine.
Oseltamivir is an effective inhibitor of influenza virus neuraminidase. Although viruses resistant to oseltamivir emerge less frequently than those resistant to amantadine or rimantadine, information on oseltamivir-resistant viruses arising during clinical use of the drug in children is limited. Our aim was to investigate oseltamivir resistance in a group of children treated for influenza.
We analysed influenza A viruses (H3N2) collected from 50 children before and during treatment with oseltamivir. We sequenced the genes for neuraminidase and haemagglutinin and studied the mutant neuraminidases for their sensitivity to oseltamivir carboxylate.
We found neuraminidase mutations in viruses from nine patients (18%), six of whom had mutations at position 292 (Arg292Lys) and two at position 119 (Glu119Val), which are known to confer resistance to neuraminidase inhibitors. We also identified another mutation (Asn294Ser) in one patient. Sensitivity testing to oseltamivir carboxylate revealed that the neuraminidases of viruses that have an Arg292Lys, Glu119Val, or Asn294Ser mutation were about 10(4)-10(5)-fold, 500-fold, or 300-fold more resistant than their pretreatment neuraminidases, respectively. Oseltamivir-resistant viruses were first detected at day 4 of treatment and on each successive day of the study. More than 10(3) infectious units per mL of virus were detected in some of the patients who did not shed drug-resistant viruses, even after 5 days of treatment.
Oseltamivir-resistant mutants in children being treated for influenza with oseltamivir arise more frequently than previously reported. Furthermore, children can be a source of viral transmission, even after 5 days of treatment with oseltamivir.
The aquaglyceroporin of Plasmodium falciparum (PfAQP) is a bi-functional channel with permeability for water and solutes. Its functions supposedly are in osmotic protection of parasites and in facilitation of glycerol permeation for glycerolipid biosynthesis. Here, we show PfAQP permeability for the glycolysis-related metabolites methylglyoxal, a cytotoxic byproduct, and dihydroxyacetone, a ketotriose. AQP3, the red cell aquaglyceroporin, also passed dihydroxacetone but excluded methylglyoxal. Proliferation of malaria parasites was inhibited by methylglyoxal with an IC50 around 200 microM. Surprisingly, also dihydroxyacetone, which is an energy source in human cells, was antiproliferative in chloroquine-sensitive and resistant strains with an IC50 around 3 mM. We expressed P. falciparum glyceraldehyde 3-phosphate dehydrogenase (PfGAPDH) to examine whether it is inhibited by either carbonyl compound. Methylglyoxal did not affect PfGAPDH on incubation with 2.5 mM for 20 h. Treatment with 2.5 mM dihydroxyacetone, however, abolished PfGAPDH activity within 6 h. Aquaglyceroporin permeability for glycolytic metabolites may thus be of physiological significance.
Using HPLC a fraction of New Zealand manuka honey has been isolated, which gives rise to the non-peroxide antibacterial activity. This fraction proved to be methylglyoxal, a highly reactive precursor in the formation of advanced glycation endproducts (AGEs). Methylglyoxal concentrations in 49 manuka and 34 non-manuka honey samples were determined using a direct detection method and compared with values obtained using standard o-phenylenediamine derivatisation. Concentrations obtained using both the methods were similar and varied from 38 to 828 mg/kg.
The 1,2-dicarbonyl compounds 3-deoxyglucosulose (3-DG), glyoxal (GO), and methylglyoxal (MGO) were measured as the corresponding quinoxalines after derivatization with orthophenylendiamine using RP-HPLC and UV-detection in commercially available honey samples. Whereas for most of the samples values for 3-DG, MGO, and GO were comparable to previously published data, for six samples of New Zealand Manuka (Leptospermum scoparium) honey very high amounts of MGO were found, ranging from 38 to 761 mg/kg, which is up to 100-fold higher compared to conventional honeys. MGO was unambigously identified as the corresponding quinoxaline via photodiodearry detection as well as by means of mass spectroscopy. Antibacterial activity of honey and solutions of 1,2-dicarbonyl towards Escherichia coli (E. coli) and Staphylococcus aureus (S. aureus) were analyzed using an agar well diffusion assay. Minimum concentrations needed for inhibition of bacterial growth (minimum inhibitory concentration, MIC) of MGO were 1.1 mM for both types of bacteria. MIC for GO was 6.9 mM (E. coli) or 4.3 mM (S. aureus), respectively. 3-DG showed no inhibition in concentrations up to 60 mM. Whereas most of the honey samples investigated showed no inhibition in dilutions of 80% (v/v with water) or below, the samples of Manuka honey exhibited antibacterial activity when diluted to 15-30%, which corresponded to MGO concentrations of 1.1-1.8 mM. This clearly demonstrates that the pronounced antibacterial activity of New Zealand Manuka honey directly originates from MGO.