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

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.

An easy, rapid, and sensitive method for detection of drug-resistant influenza virus by using a sialidase fluorescent imaging probe, BTP3-Neu5Ac

“…In order to examine the optimal concentration of calcium ion and the optimal temperature for BTP3-Neu5Ac reaction, we incubated IAV or IBV with 100 μM BTP3-Neu5Ac under varying conditions of CaCl 2 concentration and temperature. Sialidase activity was compared with that in the original experimental condition, 37°C incubation for 10 min in the presence of 1.0 mM CaCl 2 , as described in our previous reports [ 36 , 41 ]. Viral sialidase activity was increased up to 1.3 fold at 50 mM CaCl 2 ( Fig 1A ).…”

“…Drug-resistant viruses can retain sialidase activity even with the amount of an NAI that inhibits sialidase activity of drug-sensitive viruses. We have reported selective detection and selective isolation of drug-resistant viruses at the levels of virus culture and infected cells by BTP3-Neu5Ac reaction in the presence of NAIs [ 41 ]. It is expected that the BTP3-based filter method will also be able to detect such resistant viruses.…”

“…In a previous report, we described a new assay method for selective detection and isolation of an NAI-resistant IAV from live infected cells by reaction with BTP3-Neu5Ac in the presence of NAIs [ 41 ]. The time, work, and dedicated apparatus involved in this cell culture-based assay make it difficult for application of BTP3-Neu5Ac to a POCT kit for detecting NAI-resistant viruses in clinical practice and in hygiene surveys.…”

Imaging of Sialidase Activity and Its Clinical Application

“…Furthermore, we showed that drug-resistant viruses can be directly isolated from the virus-infected cell population (focus) stained with BTP3-Neu5Ac in the presence of NA inhibitors. 23) …”

“…MDCK cells on a 6-well plate were inoculated with mixtures of oseltamivir-resistant 738 and oseltamivir-sensitive 838 at various ratios (%) as described previously. 23) MDCK cells were visualized with 2 mM BTP3-Neu5Ac dropped onto the overlaid agarose-containing SFM with or without 1000 nM oseltamivir after incubation at 37°C for 2 d. Fluorescent images of the plate were observed under UV irradiation at 365 nm.…”