Visualization of avian influenza virus infected cells using self-assembling fragments of green fluorescent protein

Katsushi Kanehira,Yuko Uchida,Takehiko Saito

doi:10.1016/j.ejbt.2015.08.008

Abstract

article i nfo Background: Avian influenza viruses (AIVs) are influenza A viruses which are isolated from domestic and wild birds. AIVs that include highly pathogenic avian influenza viruses (HPAIVs) are a major concern to the poultry industry because they cause outbreaks in poultry with extraordinarily high lethality. In addition, AIVs threaten human health by occasional zoonotic infection of humans from birds. Tools to visualize AIV-infected cells would facilitate the development of diagnostic tests and preventative methods to reduce the spread of AIVs. In this study, a self-assembling split-green fluorescent protein (split-GFP) system, combined with influenza virus reverse genetics was used to construct a visualization method for influenza virus-infected cells. Results: The viral nucleoprotein (NP) segment of AIV was genetically modified to co-express GFP11 of self-assembling split-GFP, and the recombinant AIV with the modified NP segment was generated by plasmid-based reverse genetics. Infection with the recombinant AIV in cultured chicken cells was visualized by transient transfection with a GFP1-10 expression vector and fluorescence was observed in the cells at 96 hours post-inoculation. Virus titer of the recombinant AIV in embryonated eggs was comparable to wild type AIV titers at 48 h post inoculation. The inserted sequence encoding GFP11 was stable for up to ten passages in embryonated eggs. Conclusions: A visualization system for AIV-infected cells using split-GFP was developed. This method could be used to understand AIV infection dynamics in cells.

Highlights

Pathogenic avian influenza virus (HPAIV) affects the poultry industry by causing large-scale disease outbreaks in many parts of the world [1]
The stability of the inserted sequence in rCY55/GFP11-2A-NP was evaluated by serial passage in embryonated eggs from the stock culture
Sequences of the stock culture and the 10th-passaged viruses were determined by RT-PCR, followed by direct sequencing with an ABI PRISM Genetic Analyzer (Life Technologies Inc.)

Summary

Background

Avian influenza viruses (AIVs) are influenza A viruses which are isolated from domestic and wild birds. Tools to visualize AIV-infected cells would facilitate the development of diagnostic tests and preventative methods to reduce the spread of AIVs. In this study, a self-assembling split-green fluorescent protein (split-GFP) system, combined with influenza virus reverse genetics was used to construct a visualization method for influenza virus-infected cells. Infection with the recombinant AIV in cultured chicken cells was visualized by transient transfection with a GFP1-10 expression vector and fluorescence was observed in the cells at 96 hours post-inoculation. Virus titer of the recombinant AIV in embryonated eggs was comparable to wild type AIV titers at 48 h post inoculation. Conclusions: A visualization system for AIV-infected cells using split-GFP was developed. This method could be used to understand AIV infection dynamics in cells

Introduction

Construction of recombinant virus and GFP1-10 expression vector

Proliferation in embryonated chicken eggs

Results and discussion