High-Efficiency Capture of Drug Resistant-Influenza Virus by Live Imaging of Sialidase Activity.

Yuuki Kurebayashi,Tatsuya Yokozawa,Tadanobu Takahashi,Kiyoshi Ikeda,Takashi Suzuki,Nona Shibahara,Chihiro Tamoto,Hirohisa Wada,Tadamune Otsubo,Keiji Sahara,Akira Minami,Toru Takimoto

doi:10.1371/journal.pone.0156400

Abstract

Influenza A and B viruses possess a neuraminidase protein that shows sialidase activity. Influenza virus-specific neuraminidase inhibitors (NAIs) are commonly used for clinical treatment of influenza. However, some influenza A and B viruses that are resistant to NAIs have emerged in nature. NAI-resistant viruses have been monitored in public hygiene surveys and the mechanism underlying the resistance has been studied. Here, we describe a new assay for selective detection and isolation of an NAI-resistant virus in a speedy and easy manner by live fluorescence imaging of viral sialidase activity, which we previously developed, in order to achieve high-efficiency capture of an NAI-resistant virus. An NAI-resistant virus maintains sialidase activity even at a concentration of NAI that leads to complete deactivation of the virus. Infected cells and focuses (infected cell populations) of an oseltamivir-resistant virus were selectively visualized by live fluorescence sialidase imaging in the presence of oseltamivir, resulting in high-efficiency isolation of the resistant viruses. The use of a combination of other NAIs (zanamivir, peramivir, and laninamivir) in the imaging showed that the oseltamivir-resistant virus isolated in 2008 was sensitive to zanamivir and laninamivir but resistant to peramivir. Fluorescence imaging in the presence of zanamivir also succeeded in selective live-cell visualization of cells that expressed zanamivir-resistant NA. Fluorescence imaging of NAI-resistant sialidase activity will be a powerful method for study of the NAI resistance mechanism, for public monitoring of NAI-resistant viruses, and for development of a new NAI that shows an effect on various NAI-resistant mutations.

Highlights

Influenza viruses circulate worldwide and can affect every year up to 10% of the world’s population [1]
We have reported that zanamivir, one of influenza A virus- and B virus-specific NA inhibitors (NAIs), inhibited live-cell fluorescence imaging of influenza A virus-infected cells for BTP3-Neu5Ac usage [29]
We have developed a novel sialidase substrate, BTP3-Neu5Ac, for sensitive, rapid, and easy fluorescence imaging of cells infected with influenza A and B viruses

Summary

Introduction

Influenza viruses circulate worldwide and can affect every year up to 10% of the world’s population [1]. Several countries possess stockpiles of NAIs because of their effectiveness and for preparation in prevention of and treatment of patients in an influenza pandemic [6,7,8,9]. Resistance against NAIs, such as oseltamivir and zanamivir, had not been thought to be an important problem in clinical treatment because the resistance mutations reduce sialidase activity of NA to some extent [12,13,14,15,16]. Additional amino acid alterations (R222Q and V234M), which increased both sialidase activity and surface expression of NA, conferred ability of efficient virus replication, allowing worldwide spread of the oseltamivir-resistant H1N1 virus [21]. A large epidemic or pandemic of an NAI-resistant virus might have serious effects on societies and economies worldwide. Establishment of highly efficient methods for detection and isolation of NAI-resistant viruses will greatly contribute to rapid and large-scale collection of epidemiological NAI-resistance information and to studies aimed at elucidation of the NAI resistance mechanism

Methods

Results

Conclusion