Partial and Full PCR-Based Reverse Genetics Strategy for Influenza Viruses

Hongjun Chen,Matthew Angel,Daniel R Perez,Troy Sutton,Jianqiang Ye,Kemin Xu,Hongxia Shao,Andrea Ferrero,Earl G Brown

doi:10.1371/journal.pone.0046378

Abstract

Since 1999, plasmid-based reverse genetics (RG) systems have revolutionized the way influenza viruses are studied. However, it is not unusual to encounter cloning difficulties for one or more influenza genes while attempting to recover virus de novo. To overcome some of these shortcomings we sought to develop partial or full plasmid-free RG systems. The influenza gene of choice is assembled into a RG competent unit by virtue of overlapping PCR reactions containing a cDNA copy of the viral gene segment under the control of RNA polymerase I promoter (pol1) and termination (t1) signals – herein referred to as Flu PCR amplicons. Transfection of tissue culture cells with either HA or NA Flu PCR amplicons and 7 plasmids encoding the remaining influenza RG units, resulted in efficient virus rescue. Likewise, transfections including both HA and NA Flu PCR amplicons and 6 RG plasmids also resulted in efficient virus rescue. In addition, influenza viruses were recovered from a full set of Flu PCR amplicons without the use of plasmids.

Highlights

Type A Influenza (Flu) viruses belong to the family Orthomyxoviridae and their genome consist of eight segments of single strand RNA of negative polarity [1,2,3]
In order to determine whether a Flu PCR amplicon could be transfected into cells and be amplified by the influenza polymerase complex, a PCR product was produced encoding the GFP reporter gene in negative orientation flanked by the influenza segment 7 untranslated regions (UTRs) and further flanked by the human pol1 promoter and the mouse t1 termination signal, pol1EGFPt1 (Fig. 1A, Fig. S1A, Table S1)
The fluorescence signal of another amplicon, pol1EGFPutr, which lacks the t1 signal, was present in fewer cells compared to the pol1EGFPt1 amplicon indicating that run off transcription by the RNA pol1 complex may result in viral RNA (vRNA) fragments with incorrect and/or incomplete influenza sequences (Fig. 2C)

Summary

Introduction

Type A Influenza (Flu) viruses belong to the family Orthomyxoviridae and their genome consist of eight segments of single strand RNA of negative polarity [1,2,3]. RG systems for influenza rely invariably on a dual promoter concept: One for the synthesis of vRNA segments and another for the synthesis of viral mRNAs [11]. Since the termini of influenza vRNAs are crucial for virus replication, plasmids carrying a RNA polymerase I (pol1) or T7 RNA polymerase promoters have been used to generate vRNAs with the exact 39 end, whereas a pol terminator sequence (t1) or a hepatitis h ribozyme have been used to generate the exact 59 end. Plasmids carrying typical RNA polymerase II (pol2) promoters (CMV and/or chicken b-actin promoters) have been utilized for the synthesis of influenza mRNAs [15,16,17,18,19]. Flu PCR amplicons, instead of plasmids, are an efficient and viable alternative to the plasmid-based RG system

Methods

Results

Conclusion