Original Article
Biomedical
In Vitro Evaluation of Synergistic Inhibitory Effects of Neuraminidase Inhibitors and Methylglyoxal Against Influenza Virus Infection

https://doi.org/10.1016/j.arcmed.2014.12.002Get rights and content

Background and Aims

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.

Methods

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.

Results

MGO inhibited influenza virus A/WSN/33 replication 50% inhibitory concentration = 240 ± 190 μM; 50% cytotoxic concentration = 1.4 ± 0.4 mM; 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 ∼1/100th of their single usage. A synergistic effect of MGO and oseltamivir was also observed against oseltamivir-resistant virus.

Conclusions

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.

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)

  • S. Pavlovic-Djuranovic et al.

    Dihydroxyacetone and methylglyoxal as permeants of the Plasmodium aquaglyceroporin inhibit parasite proliferation

    Biochim Biophys Acta

    (2006)
  • M. Samson et al.

    Influenza virus resistance to neuraminidase inhibitors

    Antiviral Res

    (2013)
  • M. Haidari et al.

    Pomegranate (Punica granatum) purified polyphenol extract inhibits influenza virus and has a synergistic effect with oseltamivir

    Phytomedicine

    (2009)
  • H. Kawano et al.

    Genetic analysis and phylogenetic characterization of pandemic (H1N1) 2009 influenza viruses that found in Nagasaki, Japan

    Jpn J Infect Dis

    (2011)
  • T. Watanabe et al.

    Characterization of H7N9 influenza A viruses isolated from humans

    Nature

    (2013)
  • P.M. Colman

    Influenza virus neuraminidase: structure, antibodies, and inhibitors

    Protein Sci

    (1994)
  • S. Kubo et al.

    Laninamivir prodrug CS-8958, a long-acting neuraminidase inhibitor, shows superior anti-influenza virus activity after a single administration

    Antimicrob Agents Chemother

    (2010)
  • Cited by (17)

    • Fighting against the second wave of COVID-19: Can honeybee products help protect against the pandemic?

      2021, Saudi Journal of Biological Sciences
      Citation 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 Pharmaceutics
      Citation 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).

    View all citing articles on Scopus

    These authors contributed equally to this work.

    View full text