A Mechanism Underlying Attenuation of Recombinant Influenza A Viruses Carrying Reporter Genes.

Xiujuan Zhao,Yanyan Wang,Yong Yang,Lin Wang,Ping Li,Yingying Zhang,Qinghua Cui,Lijun Rong,Ruikun Du

doi:10.3390/v10120679

Abstract

Influenza A viruses (IAV) carrying reporter genes provide a powerful tool to study viral infection and pathogenesis in vivo, however, incorporating a non-essential gene into the IAV genome often results in virus attenuation and genetic instability. Very few studies have systematically compared different reporter IAVs, and most optimization attempts seem to lack authentic directions. In this study, we evaluated the ratio of genome copies to the number of infectious unit of two reporter IAVs, PR8-NS1-Gluc and PR8-PB2-Gluc. As a result, PR8-NS1-Gluc and PR8-PB2-Gluc produced 41.4 and 3.8 genomes containing noninfectious particles respectively for every such particle produced by parental PR8 virus. RdRp assay demonstrated that modification of segment NS by inserting reporter genes can interfere with the replication competitive property of the corresponding vRNAs, and the balance of the 8 segments of the reporter IAVs were drastically impaired in infected cells. As a consequence, large amounts of NS-null noninfectious particles were produced during the PR8-NS1-Gluc packaging. In summary, we unravel a mechanism underlying attenuation of reporter IAVs, which suggests a new approach to restore infectivity and virulence by introducing extra mutations compensating for the impaired replication property of corresponding segments.

Highlights

Influenza A virus (IAV) is a major cause of respiratory illness in humans, accounting for up to500,000 annual deaths worldwide [1]
We investigated the mechanism for attenuation of PR8-NS1-Gaussia luciferase (Gluc), and compared it to another reporter Influenza A viruses (IAV) PR8-Gluc which was constructed using an updated strategy [24]
Reporter IAVs PR8-NS1-Gluc and PR8-PB2-Gluc were generated by two different strategies

Summary

Introduction

Influenza A virus (IAV) is a major cause of respiratory illness in humans, accounting for up to500,000 annual deaths worldwide [1]. IAV belongs to the Orthomyxoviridae family of enveloped viruses, and its genome contain 8 single-stranded, negative-sense RNA segments (vRNAs), coding for at least 11 proteins [2]. Viruses 2018, 10, 679 after the main proteins they encode, including PB2 (Polymerase basic 2), PB1 (Polymerase basic 1), PA (Polymerase acid), HA (Hemagglutinin), NP (Nucleoprotein), NA (Neuraminidase), M (Matrix) and NS (Non-structural) [2]. The NCRs differ in length and in sequence between vRNAs, the first 13 nts at the 5’ end and the first 12 nts at the 3’ end are highly conserved, partially complementary and segment-independent [5], imposing a panhandle structure which is critical for initiation of transcription and replication [6]

Methods

Results

Conclusion