Original ArticleBiomedicalIn Vitro Evaluation of Synergistic Inhibitory Effects of Neuraminidase Inhibitors and Methylglyoxal Against Influenza Virus Infection
Introduction
Influenza viruses are enveloped, negative-stranded RNA viruses with eight segmented genomes belonging to the Orthomyxoviridae family. Two types of the influenza virus, A and B, cause influenza in humans. Influenza A viruses easily mutate, often resulting in the emergence of new antigenic variant subtypes. The threat of a human influenza pandemic has greatly increased over the past 18 years. Highly pathogenic avian influenza viruses, notably the H5N1 virus, emerged in 1997 (1). The 2009 pandemic H1N1 virus quickly spread worldwide (2) and, more recently, human infection with avian influenza H7N9 virus has been reported (3). These outbreaks should serve as warnings to responsible agencies to prepare for the next pandemic threat. At present, two main classes of anti-influenza viral drugs are available: M2 ion channel inhibitors (amantadine and rimantadine) and neuraminidase (NA) inhibitors (zanamivir, oseltamivir, laninamivir, and peramivir). The main drawbacks of M2 inhibitors are the rapid development of drug-resistant variants and inefficacy against influenza B virus 4, 5, 6. NA inhibitors were developed because of the genetic stability of the NA enzymatic active center among influenza viruses (7). NA has become a promising target for the development of antiviral drugs 8, 9. However, influenza viruses have mutated to become resistant to some NA inhibitors, resulting in decreased efficacy of these drugs 10, 11. Drug-resistant influenza viruses triggered a serious problem worldwide. For this reason, many researchers are now focused on the development of new anti-influenza treatments (12) or combination therapies to enhance the efficacy of anti-influenza viral drugs (13).
Natural products such as microbial metabolites and medicinal plants offer great promise as potentially effective and novel antiviral drugs. To date, several agents isolated from these natural products have been reported. We recently reported that manuka honey, a monofloral honey produced from the nectar of the manuka tree indigenous to New Zealand and Australia, exhibited the highest anti-influenza viral activity among tested honey samples (14). The α-ketoaldehyde compound methylglyoxal (MGO; molecular weight 72.06; Figure 1A) is present in extremely high concentrations (15) and is the major determinant of the antibacterial activities of manuka honey 16, 17. Previous studies indicated that MGO has antiviral activities against foot-and-mouth disease virus (18) and Newcastle disease virus (19). Moreover, our preliminary results showed that the concentration of MGO was 20- to 160-fold higher in manuka honey than in other honey samples. Therefore, it is possible that MGO contributes to its anti-influenza viral activity. The anti-influenza viral activity of MGO was originally reported in 1957 (20) using embryonated chicken eggs. Infection of embryonated chicken eggs is a complicated process and the anti-influenza viral mechanism of action of MGO remains poorly understood. Prior to our report, few attempts have been made to elucidate the anti-influenza virus activity of MGO over the past half century.
In this study we investigated the anti-influenza viral activity of MGO and its potential as a combination treatment with NA inhibitors. We found that MGO was effective against various influenza strains, including the 2009 pandemic virus, which is resistant to oseltamivir. In addition, we evaluated the synergistic effect of NA inhibitors and MGO against influenza virus infection.
Section snippets
Cells, Viruses, and Chemicals
Madin–Darby canine kidney (MDCK) cells were grown in Eagle's minimum essential medium (E-MEM) supplemented with 5% fetal bovine serum (FBS) at 37°C in an atmosphere of 5% CO2. Influenza virus A strains A/Puerto Rico/8/34 (H1N1), A/Hong Kong/8/68 (H3N2), and A/duck/Pennsylvania/1/84 (H5N2) were propagated in MDCK cells in the presence of 2.5 μg/mL of trypsin (Sigma-Aldrich Co., St. Louis, MO). The 50% tissue culture infective dose (TCID50) of influenza virus was titrated using MDCK cells.
MGO Suppresses Influenza Virus Replication
A previous study suggested that several α-ketoaldehyde compounds, including MGO, can suppress influenza virus replication in embryonated chicken eggs (20). However, the precise quantitative evaluation of MGO such as cytotoxicity and anti-influenza viral activity has not yet been fully understood. We first evaluated the cytotoxicity of MGO against MDCK cells using the WST-1 assay and CV staining (Figure 1B). CV staining is an alternative and rapid method for the evaluation of cytotoxicity (24).
Discussion
Influenza virus is a serious threat to human health. Thus, there is an urgent requirement for the development of novel anti-influenza viral drugs. In consideration of the findings of a previous report using embryonated chicken eggs (20) and those of our recent report regarding the anti-influenza viral activity of manuka honey (14), we hypothesized that MGO is effective against various influenza viruses, including the pandemic 2009 H1N1 virus as evaluated in the present study using MDCK cells.
Acknowledgments
This work was partly supported by Yamada Research Grant (#0107 and #0131) and a grant from the gCOE Program of Nagasaki University.
Conflict of interests: There is no conflict of interest to disclose.
References (35)
- et al.
Human influenza A H5N1 virus related to a highly pathogenic avian influenza virus
Lancet
(1998) - et al.
The M2 proton channels of influenza A and B viruses
J Biol Chem
(2006) - et al.
Emergence of amantadine-resistant influenza A viruses: epidemiological study
J Infect Chemother
(2003) - et al.
Influenza virus M2 protein has ion channel activity
Cell
(1992) - et al.
Influenza virus neuraminidase inhibitors
Lancet
(2000) - et al.
Resistant influenza A viruses in children treated with oseltamivir: descriptive study
Lancet
(2004) - et al.
Anti-influenza viral effects of honey in vitro: potent high activity of manuka honey
Arch Med Res
(2014) - et al.
Isolation by HPLC and characterisation of the bioactive fraction of New Zealand manuka (Leptospermum scoparium) honey
Carbohydr Res
(2008) - et al.
Comparison of methods of interpretation of checkerboard synergy testing
Diagn Microbiol Infect Dis
(2002) - et al.
In vitro neuraminidase inhibitory activities of four neuraminidase inhibitors against influenza viruses isolated in the 2010–2011 season in Japan
J Infect Chemother
(2012)
Dihydroxyacetone and methylglyoxal as permeants of the Plasmodium aquaglyceroporin inhibit parasite proliferation
Biochim Biophys Acta
Influenza virus resistance to neuraminidase inhibitors
Antiviral Res
Pomegranate (Punica granatum) purified polyphenol extract inhibits influenza virus and has a synergistic effect with oseltamivir
Phytomedicine
Genetic analysis and phylogenetic characterization of pandemic (H1N1) 2009 influenza viruses that found in Nagasaki, Japan
Jpn J Infect Dis
Characterization of H7N9 influenza A viruses isolated from humans
Nature
Influenza virus neuraminidase: structure, antibodies, and inhibitors
Protein Sci
Laninamivir prodrug CS-8958, a long-acting neuraminidase inhibitor, shows superior anti-influenza virus activity after a single administration
Antimicrob Agents Chemother
Cited by (17)
Fighting against the second wave of COVID-19: Can honeybee products help protect against the pandemic?
2021, Saudi Journal of Biological SciencesCitation Excerpt :Profound, in vitro antiviral activity of a mixture of natural honey, ginger and garlic extracts against various strains of influenza virus was observed, moreover they showed in their study that this mixture promotes the proliferation of human lymphocytes (Vahed & Batool Jafri 2016). 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.
Aqueous solutions of didecyldimethylammonium chloride and octaethylene glycol monododecyl ether: Toward synergistic formulations against enveloped viruses
2016, International Journal of PharmaceuticsCitation Excerpt :Indeed, the infectivity titers of CVB4 is reduced only slightly after 15 min of incubation with [DiC10][Cl]/C12E8 mixture (500/500 μM or 181/269 ppm): only 1-log of CVB4 is inactivated in contrast other lipid-containing viruses present at least 7-log reduction. Therefore, the viral envelope is the target of the [DiC10][Cl]/C12E8 mixed micelles Numerous references emphasizing synergistic effects of a particular substance upon the virucidal activity of other compounds can be found in the literature (Bauer, 1955; Lavrov et al., 1968; Liu et al., 2005; Kramer et al., 2006; Hayashi et al., 2012; Charyasriwong et al., 2015). However, based on our previous work, we have highlighted that [DiC10][Cl] and C12E8 detergents neutralize each other (Rauwel et al., 2012).
Benefits of Manuka Honey in the Management of Infectious Diseases: Recent Advances and Prospects
2023, Mini-Reviews in Medicinal ChemistryLead Optimization of Influenza Virus RNA Polymerase Inhibitors Targeting PA-PB1 Interaction
2022, Journal of Medicinal ChemistryNatural bioactive compounds of honey and their antimicrobial activity
2022, Czech Journal of Food Sciences
- ∗
These authors contributed equally to this work.