Combination of specific single chain antibody variable fragment and siRNA has a synergistic inhibitory effect on the propagation of avian influenza virus H5N1 in chicken cells.

Shuang Wang,Peng Zhang,Ji Xi,Qiang Hou,Wei-Quan Liu,Ji-Gui Wang,Ya-Ping Mao,Dao-Li Yuan,Fei He,Jia-Zeng Sun,Zi-Ding Zhang,Zhi-Li Li,Bao Yi

doi:10.1186/s12985-014-0208-x

Abstract

BackgroundThe avian influenza virus (AIV) causes frequent disease with high morbidity and mortality. RNA interference (RNAi) has been shown to provide an effective antiviral defense in animals, and several studies have focused on harnessing small interfering RNAs (siRNAs) to inhibit viral infections. In addition, single chain variable fragments (scFvs) contain the complete antigen binding site, and specific scFvs can bind to and neutralize viruses.ResultsFourteen positive scFvs were selected by the yeast two-hybrid system. Using molecular docking technology, we selected the three highest affinity scFvs for further functional validation. Results of indirect ELISA and IFA showed that all three scFvs could bind to FJ13 strain and had neutralizing activity, decreasing the viral infectivity markedly. Chicken fibroblastic DF-1 cells were transfected with scFvs in combination with siRNA-NP604 (an siRNA of anti-AIV NP protein previously reported). Following infection with FJ13 virus, copy numbers of the virus were significantly reduced from 12 h to at least 60 h post-infection compared to that achieved in cells transfected with scFv or siRNA-NP604 separately.ConclusionsA novel combination of antiviral siRNAs expressed in chicken cells and chicken antibody single-chain variable fragments (scFvs) secreted from the cells has a synergistic inhibitory effect on the avian influenza viral proliferation in vitro. Intracellular application of scFvs and anti-viral siRNA may provide a new approach to influenza prevention and treatment.

Highlights

Avian influenza viruses (AIVs) are important pathogens affecting the poultry industry worldwide, with some infecting humans with a high fatality rate [1]
We report that use of small interfering RNAs (siRNAs) targeting the NP protein, in combination with single chain variable fragments of anti-HA protein antibody, has a synergistic inhibitory effect on the propagation of avian influenza virus H5N1
Three scFvs binding to the HA protein of AIV FJ13 strain with high affinity were selected by the yeast two-hybrid system and molecular docking technology

Summary

Introduction

Avian influenza viruses (AIVs) are important pathogens affecting the poultry industry worldwide, with some infecting humans with a high fatality rate [1]. ScFvs, another effective countermeasure against animal virus, are among the most widely used recombinant antibodies (rAbs) as they have been successfully modified into a number of different Ab formats and are expressed by several expression systems. Since they contain the complete antigen binding site, specific scFvs can bind to and neutralize viruses [10,11,12]. A high-affinity human scFv antibody against the recombinant H5N1 virus hemagglutinin ectodomain (HA1) showed satisfactory antiviral effects against challenge with H5N1 viruses in embryonated chicken eggs [14]. Single chain variable fragments (scFvs) contain the complete antigen binding site, and specific scFvs can bind to and neutralize viruses

Methods

Results

Discussion

Conclusion