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

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.

Generation of a Reassortant Influenza A Subtype H3N2 Virus Expressing Gaussia Luciferase

“…In the present study, the strategy of fusing reporter genes to the NS1 open reading frame that was successfully used to construct reporter influenza A subtype H1N1 viruses [28,45] failed to generate a reporter influenza A subtype H3N2 virus (Figure 1). This is not surprising, since our previous study demonstrated that the vRNA replication of an IAV segment can be impaired by the insertion of exogenous genes, resulting in an imbalance of the eight segments during genome replication [33]. We speculate that the NS segment of IAV H3N2 is more vulnerable than that of H1N1 to the modification.…”

“…The TCID 50 values were determined using MDCK cells and the titer was calculated by the Reed–Muench method [33].…”

Screening for Anti‐Influenza Actives of Prefractionated Traditional Chinese Medicines

“…Previously, we generated a recombinant IAV expressing Gaussia luciferase, based on which a phenotypic high-throughput screening approach was subsequently established, providing a powerful tool for antiviral discovery [ 11 , 12 , 15 ]. A phenotypic screening was therefore carried out against the prefractionated TCM library for anti-influenza actives.…”

“…Madin–Darby canine kidney (MDCK) epithelial cells were grown in Dulbecco's modified Eagle's medium (DMEM; Cellgro, Manassas, VA, USA) supplemented with 10% fetal bovine serum (FBS; Gibco, Carlsbad, CA, USA), 1,000 units/mL penicillin and 100 μ g/mL of streptomycin (Invitrogen, Carlsbad, CA, USA). The replication-competent reporter influenza A virus carrying the Gaussia luciferase gene (PR8-PB2-Gluc) and wildtype influenza A/Puerto Rico/8/34 (H1N1, PR8) were propagated as previously described [ 11 , 12 , 15 ]. Infections were performed in Opti-MEM containing 1.5 µ g/mL N-tosyl-L-phenylalanine chloromethyl ketone (TPCK)-trypsin (Sigma-Aldrich, St. Louis, MO, USA).…”

A Parallel Phenotypic Versus Target-Based Screening Strategy for RNA-Dependent RNA Polymerase Inhibitors of the Influenza A Virus

“…Human embryonic kidney cell line 293T and Madin-Darby canine kidney (MDCK) epithelial cells were obtained from Dr. Fei Deng (Wuhan Institute of Virology, CAS, China) and grown in Dulbecco’s modified Eagle’s medium (DMEM; Cellgro, Manassas, VA, USA) supplemented with 10% fetal bovine serum (FBS; Gibco, Carlsbad, CA, USA), 1000 units/mL penicillin, and 100 μg/mL of streptomycin (Invitrogen, Carlsbad, CA, USA). The influenza A reporter PR8-Gluc virus and wild-type PR8 virus were generated and stocked in our lab as previously described [35].…”